C 1998 The Japan Mendel Society Cytologia 63: 329-339, 1998 A Chromosome Phylogeny of the Droseraceae by Using CMA-DAPI Fluorescent Banding Yoshikazu Hoshi and Katsuhiko Kondo1 Laboratory of Plant Chromosome and Gene Stock, Faculty of Science, Hiroshima University, Higashi-Hiroshima City 739-8526, Japan Accepted May 21, 1998 Summary Aldrovanda vesiculosa L., Dionaea muscipula Ellis, 20 species of Drosera L. and Dros- ophyllum lusitanicum Link. were investigated for a metaphase chromosome analysis by using the se- quentially fluorescent CMA and DAPI staining methods. Aldrovanda vesiculosa and the Drosera species showed numerous small-sized chromosomes and some middle-sized chromosomes with no primary constriction. Dionaea muscipula had the middle-sized chromosomes with the localized cen- tromeres and CMA-negative and DAPI-positive pericentric bands. Drosophyllum lusitanicum dis- played quite large-sized chromosomes with well-defined localized-centromeres. Computer-aided measurements of metaphase chromosomes stained with CMA and DAPI showed that Aldrovanda vesiculosa and the Drosera species displayed the common features such as localization of CMA-pos- itive and DAPI-negative satellites and non-staining region. Key words CMA, DAPI, Diffused centromere, Aldrovanda vesiculosa, Dionaea muscipula, Drosera, Drosophyllum lusitanicum, Droseraceae, Localized centromere. The Droseraceae consists of four genera; the monotypic Aldrovanda L., Dionaea Ellis and Drosophyllum Link., and the polytypic Drosera L. with approximately 90 species (Diels 1906, Marchant and George 1982). The Droseraceae is a monophyletic family with remarkable differ- ences of chromosomes in size (Rothfels and Heimburger 1968, Stebbins 1971) and in localized and diffused centromeres (Behre 1929, Rothfels and Heimburger 1968, Kondo et al. 1976, Kondo and Segawa 1988, Sheikh and Kondo 1995, Sheikh et al. 1995, Hoshi and Kondo 1998). Drosera has a diffused centromeric chromosome which is disadvantage to designate conventional karyotype (Kondo et al. 1976). Some species of Drosera have a difficulty of chromosome measurements due to small-sized chromosome with obscure euchromatic ends (Rothfels and Heimburger 1968). Extensive cytological studies have been done in Aldrovanda vesiculosa (Kondo 1969, Kress 1970), Dionaea muscipula (Behre 1929, Sato 1947, Kondo 1970), various species of Drosera (e.g. Shimamura 1941, Wood 1955, Kondo 1966, 1969, 1970, 1973, 1976, 1984, Kondo et al. 1976, Kondo and Olivier 1979, Ficini et al. 1980, Beuzenberg and Hair 1983, Kondo and Segawa 1988, Hoshi et al. 1994, Kondo et al. 1994, Sheikh and Kondo 1995, Sheikh et al. 1995, Hoshi and Kondo 1998), and Drosophyllum lusitanicum (Behre 1929, Rothfels and Heimburger 1968). Recently some Drosera studies have demonstrated that fluorochromes such as chromomycin A3 (CMA) and 4',6-diamidino-2-phenylindole (DAPI) were useful dyes to obtain well-stained chromosomes with clear ends and characteristic bands (Sheikh and Kondo 1995, Hoshi and Kondo 1998). Furthermore, computer-aided metaphase measurements offered objective and accurate chro- mosomal analysis (Hoshi and Kondo 1998). In this study, CMA and DAPI fluorescent staining karyotypes in Aldrovanda vesiculosa, Dion- aea muscipula, 20 species of Drosera, and Drosophyllum lusitanicum are analyzed by using a com- 1 Corresponding author: E-mail: [email protected] Contribution no. 58 of Laboratory of Plant Chro- mosome and Gene Stock. 330 Yoshikazu Hoshi and Katsuhiko Kondo Cytologia 63 puter to justify phylogenetic relationships and to speculate speciation and evolution in chromo- somes of the Droseraceae. Materials and methods Plant materials Aldrovanda vesiculosa, Dionaea muscipula, 20 species of Drosera and Drosophyllum lusitan- icum used are listed in Table 1. Seeds of the Drosera species were purchased from Western Aus- tralia, Australia and sowed and germinated both in vitro and in vivo in the Laboratory of Plant Chromosome and Gene Stock, Faculty of Science, Hiroshima University. For in vitro culture of the Drosera species seeds were surface-sterilized and sown on sterilized 1/2 strength of Murashige and Skoog basal medium (MS; Murashige and Skoog 1962) supplemented with 0.2% gelrite without Table 1. Source of the materials of Aldrovanda vesiculosa, Dionaea muscipula, 20 species of Drosera and Drosophyllum lusitanicum investigated 1998 Chromosome Phylogeny of the Droseraceae 331 any growth substance. Taxonomic treatment of Drosera followed basically Diels (1906). Slide preparation Growing root tips in vitro were harvested and pretreated with 0.002 M hydroxyquinoline for 2 hr at 18C before fixation with 45% acetic acid for 5 min and then, hydrolysis with a mixture of 1N hydrochloric acid and 45% acetic acid (2 : 1) at 60C for 7 sec. Their root meristems were cut and squashed in 45% acetic acid. The preparations were air-dried for 48 hr at room temperature after removal of coverslips with dry ice. Fluorescent staining The method of sequentially fluorescent staining with distamycin A and chromomycin A3 (DMA-CMA staining) followed Schweizer (1981) with slight modification. The air-dried slides after the coverslip was removed were used for sequential DMA-CMA staining: They were preincu- bated in Mcllvine's buffer (pH 7.0) for 30 min, treated with 0.1 mg/ml DMA (Sigma) in Mcllvine's buffer for 10 min in a humid chamber and rinsed in Mcllvine's buffer supplemented with 2.5 mg/ml MgSO4 for 10 min. After rinsed, they were stained with 0.1 mg/ml CMA (Sigma) in Mcllvine's buffer supplemented with Mg504 for 10 min. Then, they were mounted with glycerol and stored at 4C for 12 hr to prevent fading. These chromosomes stained with DMA-CMA were irradiated with BV (blue violet) filter cassette and fluoresced yellow. The method of sequentially fluorescent staining with actinomycin D and DAPI (AMD-DAPI staining) followed Schweizer (1976) with slight modification. After DMA-CMA staining, the slides were used for sequential AMD-DAPI staining: The slides were destained in 45% acetic acid for 30 min, rinsed in distilled water for 5 min. They were dipped in Mcllvine's buffer for 30 min, treated in 0.25 mg/ml AMD (Sigma) in Mcllvine's buffer for 15 min in a humid chamber and rinsed in Mcllvine's buffer for 10 min. After rinsed, they were mounted in glycerol and observed immediate- ly. These chromosomes stained with AMD-DAPI were irradiated with UV (ultra violet) filter cas- sette and fluoresced blue. Photographs were taken with the Professional T-MAX film (ISO 400) in a Nikon fluorescent microscope. Chromosome measurements Chromosome classification followed Hoshi and Kondo (1998). Additionally, super large-sized chromosomes of Drosophyllum lusitanicum were designated karyotype formulae. The karyotype formulae were based on measurements of 30 metaphase cells. Chromosome sizes were defined as: Super large (LL) >15.00 um2 in area, large (L) 6.00-14.99 um2, middle (M) 1.50-5.99 pm2, and small (5) <1.49 um2. Degree of karyotype symmetry at mitotic metaphase was estimated according to Stebbins' classification (1971). Ratio of the largest chromosome to the smallest chromosome was calculated by average area of the largest chromosome/average area of the smallest chromosome. In- terchromosomal asymmetry index proposed by Romero Zarco (1986) was calculated by standard deviation of average area of chromosome complement/average area of chromosome complement. This index provides a way of estimating variation of chromosome area. Results and discussion Figs. 1-3 illustrate the results of DMA-CMA and AMD-DAPI staining in the chromosomes of Aldrovanda vesiculosa, Dionaea muscipula, 20 species of Drosera, and Drosophyllum lusitanicum of the Droseraceae. Table 2 and Fig. 4 show the results of mitotic chromosome numbers, total chro- mosome area and standard deviation, individual chromosome area from the largest to the smallest chromosome, average area of chromosome complement+ standard deviation of each species and 332 Yoshikazu Hoshi and Katsuhiko Kondo Cytologia 63 1998 Chromosome Phylogeny of the Droseraceae 333 334 Yoshikazu Hoshi and Katsuhiko Kondo Cytologia 63 Fig. I. Mitotic-metaphase chromosomes in somatic cells of Drosera, subgen. Rorella, sect. Rossolis stained with DMA-CMA (a, c, e, g, i, k, m, o, q) and AMD-DAPI (b, d, f, h, j, 1, n, p, r). a, b) D. aliciae. c, d) D. capensis. e, f) D. collinsiae. g, h) D. dielsiana. i, j) D. hilaris. k, 1) D. madagascariensis. m, n) D. montana. o, p) D. trinervia. Two middle-sized chromosomes were observed (arrowheads). q, r) D. vil- losa. CMA-positive and DAPI-negative sites were observed in D. aliciae, D. capensis, D. dielsiana, D. hilaris, D. madagascariensis, D. montana, D. trinervia and D. villosa (arrows). Bar= 5 pm. 1998 Chromosome Phylogeny of the Droseraceae 335 Fig. 2. Mitotic-metaphase chromosomes in somatic cells of Drosera stained with DMA-CMA (a, c, e, g, i, k, m, o, q, s, u) and AMD-DAPI (b, d, f, h, j, 1, n, p, r, t, v). a, b) D. burmanni, sect. Thelocalyx. c, d) D. sessilifolia, sect. Thelocalyx. e, f) D. adelae, sect. Arachnopus. g, h) D. indica, sect. Arachnopus. i,j) D. prolifera, sect. Arachnopus. k, l) D. hamiltonii, sect. Stelogyne. m, n) D. binata, sect. Phycopsis. o, p) D. cist(ora, sect. Ptycnostigma, q, r) D. pauciflora, sect. Ptycnostigma. Two middle- and six small-sized chromosomes (arrowheads) were more than 1.00 pm2 area, while 32 small-size chromosomes less than 0.99 um2 area. s, t) D. auriculata, sect. Polypeltes. u, v) D. peltata, sect. Polypeltes. CMA-positive and DAPI-negative sites were observed in D. burmanni, D. sessilifolia, D. adelae, D. indica, D. prolifera, D. hamiltanii, D. binata, D. cistiflora, D. pauciflora and D. peltata (arrows). Bar=5 pm. Cytologia 63 336 Yoshikazu Hoshi and Katsuhiko Kondo Fig. 3. Mitotic-metaphase chromosomes in somatic cells of three monotypic genera of the Droser- aceae stained with DMA-CMA (a, c, e) and AMD-DAPI (b, d, f). a, b) Aldrovanda vesiculosa. The chro- mosomes did not obviously visualize any localized-centromere and showed CMA-positive and DAPI- negative sites (arrows).