© 2010 The Japan Mendel Society Cytologia 75(3): 255–260, 2010 Cytogeography of Glechoma hederacea subsp. grandis (Labiatae) in Japan Norihito Miura and Yoshikane Iwatsubo* Graduate School of Science and Engineering, University of Toyama, Gofuku 3190, Toyama 930–8555, Japan Received February 26, 2010; accepted August 28, 2010 Summary In this study, we examined the chromosomal number for Glechoma hederacea subsp. grandis in a total of 1,030 specimens collected from different distribution areas in Japan. We found that G. hederacea subsp. grandis could be categorized into 3 cytotypes with 2nϭ36 (tetraploid), 2nϭ45 (pentaploid) and 2nϭ54 (hexaploid) chromosomes. Tetraploid plants were found throughout different areas in Japan; however, hexaploid plants were mainly distributed in central Honshu, Shikoku and Kyushu. Likewise, pentaploid plant distribution was found to overlap with hexaploid plant distribution areas. The pentaploid plant group appeared only in regions common to both tetraploid and hexaploid plants. All 3 cytotypes were found to have karyotypes which could be represented by the following equations: A) 6Mϩ4mϩ18smϩ8st for tetraploids, B) 6Mϩ15mϩ19smϩ5st for pentaploids, and C) 6Mϩ26mϩ20smϩ2st for hexaploids. Pentaploid specimen karyotypes had half the tetraploid and half the hexaploid chromosomal set, indicating that this specimen was a hybrid between tetraploid and hexaploid plants. Key words Geographic distribution, Glechoma hederacea subsp. grandis, Hybrid, Karyotype, Polyploidy. Glechoma L. (Labiatae), distributed across north temperate zones in Eurasia, is a small genus with 4 to 8 species (Budantsev 2004). One of its species, G. hederacea L., has a wide distribution range occurring spontaneously throughout Eurasia. Furthermore, this species can be divided into subsp. hederacea distributed in Europe, and subsp. grandis (A. Gray) H. Hara distributed in Far East Asia (Hara et al. 1954). The subsp. grandis (Fig. 1) is the only taxon of this genus found in Japan and known to have a chromosomal count of 2nϭ36 (Tanaka 1953, Hara et al. 1954, Nishikawa 1985). However, Iwatsubo et al. (2004) revealed that G. hederacea subsp. grandis located in Toyama Prefecture, situated in the central region of Honshu in Japan, had 3 cytotypes corresponding to 2nϭ36, 45 and 54, and interpreted these as tetraploids, pentaploids, and hexaploids, respectively. Moreover, the authors concluded Fig. 1. Photograph of plant of Glechoma hederacea that of the 3 chromosomal forms, the pentaploid subsp. grandis. * Corresponding author, e-mail: [email protected] 256 N. Miura and Y. Iwatsubo Cytologia 75(3) form was a hybrid between tetraploid and hexaploid plants (Iwatsubo et al. 2004). A recent study by Miura and Iwatsubo (2008) found that G. hederacea subsp. grandis was also distributed in Miyagi Prefecture, the Tohoku districts of Honshu. Their study also revealed that most plants were tetraploids, with only a fraction of hexaploid plants. The studies conducted from the 2 prefectures indicated that each chromosomal form of G. hederacea subsp. grandis could have different distributions in Japan. Therefore, we decided to study the distribution and karyotype of the 3 cytotypes of this subspecies in order to shed light on the putative origin of the pentaploid plant. Materials and methods A total of 1,030 specimens of G. hederacea L. subsp. grandis were collected from 945 localities in Japan. Newly formed adventitious root tips were collected and pretreated with 2 mM 8- hydroxyquinoline solution at room temperature for 1–1.5 h, and maintained at approximately 5°C for 15 h. The root tips were then fixed with a mixture of glacial acetic acid and absolute ethyl alcohol (1 : 3), and incubated at room temperature for 1 h. The root tips were then macerated in 1 N hydrochloric acid at 60°C for 10 min, and washed in tap water. Finally, the samples were stained and squashed in a drop of 1.5% lacto-propionic orcein on a glass slide, and fully-spread metaphase chromosomes were examined. Collection localities for the specimens were as follows: A) tetraploid plants from Higashikuromaki, Toyama City, Toyama Prefecture, B) pentaploid plants from Hio, Toyama City, Toyama Prefecture, and C) hexaploid plants from Monjuji, Toyama City, Toyama Prefecture. Chromosomal forms were expressed according to the nomenclature outlined by Levan et al. (1964). Voucher specimens of the plants examined have been deposited at the Toyama Science Museum (TOYA). Results and discussion Chromosome numbers Chromosomal counts determined in this study for G. hederacea subsp. grandis were based on 1,030 specimens collected from the distribution areas of Japan known to have the 3 cytotypes (2nϭ36: tetraploids, 2nϭ45: pentaploids, 2nϭ54: hexaploids) of this taxon (Fig. 2). These distribution areas almost corresponded to previous reports for this taxon in Japan (Tanaka 1953, Hara et al. 1954, Nishikawa 1985, Iwatsubo et al. 2004, Miura and Iwatsubo 2008). From a total of 1,030 specimens, 817 (79.3% of the plants examined) were tetraploids with 2nϭ36 chromosomes, 83 (8.1%) were pentaploids with 2nϭ45 and 130 (12.6%) were hexaploids with 2nϭ54. Fig. 2. Photographs of somatic metaphase chromosomes of 3 chromosome forms of Glechoma hederacea subsp. grandis, A: 2nϭ36; B: 2nϭ45; C: 2nϭ54. Bar indicates 5 mm. 2010 Cytogeography of Glechoma hederacea subsp. grandis (Labiatae) in Japan 257 Fig. 3. Distribution of area of the 3 chromosome forms of Glechoma hederacea subsp. grandis in Japan, A: 2nϭ36; B: 2nϭ45; C: 2nϭ54. Bar indicates 200 km. Fig. 4. Karyotypes of somatic metaphase of 3 chromosome forms of Glechoma hederacea subsp. grandis, A: 2nϭ36; B: 2nϭ45; C: 2nϭ54. Bar indicates 5 mm. 258 N. Miura and Y. Iwatsubo Cytologia 75(3) Distribution areas for the 3 cytotypes were as follows: A) Tetraploids (Fig. 3A) occurred throughout Hokkaido, Honshu, Shikoku, and Kyushu. In Hokkaido, all plants were tetraploids. Almost all plants located in the northern part of Honshu (Tohoku and Kanto districts) were also tetraploid. B) Pentaploids (Fig. 3B) were distributed in central Honshu, Shikoku and Kyushu. However, this plant occurred only in the distribution area common to the hexaploid plant. C) Hexaploids occurred mainly in central Honshu, Shikoku and Kyushu (Fig. 3C). Karyotype analysis Table 2. Masurements of somatic metaphase chromosomes of 2nϭ45 Glechoma hederacea Tetraploid plants had chromosomes at subsp. grandis metaphase which ranged from 0.9 mm to Chromosome 2.2 mm in length, and had an arm ratio between No. Length (mm) Arm ratio 1.0 and 4.5 (Fig. 4A). As shown in Table 1, the form 36 chromosomes were divided into 3 groups: 1 0.3ϩ1.4ϭ1.7 4.7 st 2 0.3ϩ1.3ϭ1.6 4.3 st Table 1. Measurements of somatic metaphase chromo- 3 0.4ϩ1.2ϭ1.6 3.0 sm somes of 2nϭ36 Glechoma hederacea subsp. 4 0.4ϩ1.2ϭ1.6 3.0 sm grandis 5 0.4ϩ1.2ϭ1.6 3.0 sm 6 0.5ϩ1.1ϭ1.6 2.2 sm Chromosome 7 0.5ϩ1.1ϭ1.6 2.2 sm No. Length (mm) Arm ratio form 8 0.6ϩ1.0ϭ1.6 1.7 m 9 0.6ϩ0.9ϭ1.5 1.5 m 1 0.4ϩ1.8ϭ2.2 4.5 st 10 0.6ϩ0.9ϭ1.5 1.5 m 2 0.4ϩ1.7ϭ2.1 4.3 st 11 0.3ϩ1.2ϭ1.5 4.0 st 3 0.4ϩ1.5ϭ1.9 3.8 st 12 0.3ϩ1.2ϭ1.5 4.0 st 4 0.4ϩ1.5ϭ1.9 3.8 st 13 0.3ϩ1.1ϭ1.4 3.7 st 5 0.4ϩ1.4ϭ1.8 3.5 st 14 0.4ϩ1.0ϭ1.4 2.5 sm 6 0.4ϩ1.4ϭ1.8 3.5 st 15 0.4ϩ1.0ϭ1.4 2.5 sm 7 0.4ϩ1.4ϭ1.8 3.5 st 16 0.4ϩ1.0ϭ1.4 2.5 sm 8 0.4ϩ1.4ϭ1.8 3.5 st 17 0.5ϩ0.9ϭ1.4 1.8 sm 9 0.5ϩ1.3ϭ1.8 2.6 sm 18 0.5ϩ0.8ϭ1.3 1.6 m 10 0.5ϩ1.3ϭ1.8 2.6 sm 19 0.6ϩ0.7ϭ1.3 1.2 m 11 0.5ϩ1.2ϭ1.7 2.4 sm 20 0.6ϩ0.7ϭ1.3 1.2 m 12 0.5ϩ1.2ϭ1.7 2.4 sm 21 0.3ϩ0.9ϭ1.2 3.0 sm 13 0.4ϩ1.2ϭ1.6 3.0 sm 22 0.3ϩ0.9ϭ1.2 3.0 sm 14 0.4ϩ1.2ϭ1.6 3.0 sm 23 0.4ϩ0.8ϭ1.2 2.0 sm 15 0.5ϩ0.9ϭ1.4 1.8 sm 24 0.4ϩ0.8ϭ1.2 2.0 sm 16 0.5ϩ0.9ϭ1.4 1.8 sm 25 0.4ϩ0.8ϭ1.2 2.0 sm 17 0.7ϩ0.7ϭ1.4 1.0 M 26 0.4ϩ0.8ϭ1.2 2.0 sm 18 0.7ϩ0.7ϭ1.4 1.0 M 27 0.5ϩ0.7ϭ1.2 1.4 m 19 0.7ϩ0.7ϭ1.4 1.0 M 28 0.5ϩ0.7ϭ1.2 1.4 m 20 0.7ϩ0.7ϭ1.4 1.0 M 29 0.5ϩ0.7ϭ1.2 1.4 m 21 0.5ϩ0.8ϭ1.3 1.6 m 30 0.5ϩ0.7ϭ1.2 1.4 m 22 0.5ϩ0.8ϭ1.3 1.6 m 31 0.5ϩ0.6ϭ1.1 1.2 m 23 0.4ϩ0.9ϭ1.3 2.3 sm 32 0.4ϩ0.7ϭ1.1 1.8 sm 24 0.4ϩ0.9ϭ1.3 2.3 sm 33 0.4ϩ0.7ϭ1.1 1.8 sm 25 0.6ϩ0.7ϭ1.3 1.2 m 34 0.3ϩ0.7ϭ1.0 2.3 sm 26 0.6ϩ0.7ϭ1.3 1.2 m 35 0.3ϩ0.7ϭ1.0 2.3 sm 27 0.6ϩ0.6ϭ1.2 1.0 M 36 0.4ϩ0.6ϭ1.0 1.5 m 28 0.6ϩ0.6ϭ1.2 1.0 M 37 0.5ϩ0.5ϭ1.0 1.0 M 29 0.3ϩ0.8ϭ1.1 2.7 sm 38 0.5ϩ0.5ϭ1.0 1.0 M 30 0.3ϩ0.8ϭ1.1 2.7 sm 39 0.5ϩ0.5ϭ1.0 1.0 M 31 0.4ϩ0.7ϭ1.1 1.8 sm 40 0.5ϩ0.5ϭ1.0 1.0 M 32 0.4ϩ0.7ϭ1.1 1.8 sm 41 0.4ϩ0.5ϭ0.9 1.3 m 33 0.4ϩ0.7ϭ1.1 1.8 sm 42 0.4ϩ0.5ϭ0.9 1.3 m 34 0.4ϩ0.7ϭ1.1 1.8 sm 43 0.4ϩ0.4ϭ0.8 1.0 M 35 0.3ϩ0.7ϭ1.0 2.3 sm 44 0.4ϩ0.4ϭ0.8 1.0 M 36 0.3ϩ0.6ϭ0.9 2.0 sm 45 0.3ϩ0.4ϭ0.7 1.3 m 2010 Cytogeography of Glechoma hederacea subsp.