© 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 2n 36 (tetraploid), 2n 45 (pentaploid) and 2n 54 (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) 6M 4m 18sm 8st for tetraploids, B) 6M 15m 19sm 5st for pentaploids, and C) 6M 26m 20sm 2st 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 2n 36 (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 2n 36, 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 (2n 36: tetraploids, 2n 45: pentaploids, 2n 54: 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 2n 36 chromosomes, 83 (8.1%) were pentaploids with 2n 45 and 130 (12.6%) were hexaploids with 2n 54.
Fig. 2. Photographs of somatic metaphase chromosomes of 3 chromosome forms of Glechoma hederacea subsp. grandis, A: 2n 36; B: 2n 45; C: 2n 54. 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: 2n 36; B: 2n 45; C: 2n 54. Bar indicates 200 km.
Fig. 4. Karyotypes of somatic metaphase of 3 chromosome forms of Glechoma hederacea subsp. grandis, A: 2n 36; B: 2n 45; C: 2n 54. 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 2n 45 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 0.3 1.4 1.7 4.7 st 2 0.3 1.3 1.6 4.3 st Table 1. Measurements of somatic metaphase chromo- 3 0.4 1.2 1.6 3.0 sm somes of 2n 36 Glechoma hederacea subsp. 4 0.4 1.2 1.6 3.0 sm grandis 5 0.4 1.2 1.6 3.0 sm 6 0.5 1.1 1.6 2.2 sm Chromosome 7 0.5 1.1 1.6 2.2 sm No. Length (mm) Arm ratio form 8 0.6 1.0 1.6 1.7 m 9 0.6 0.9 1.5 1.5 m 1 0.4 1.8 2.2 4.5 st 10 0.6 0.9 1.5 1.5 m 2 0.4 1.7 2.1 4.3 st 11 0.3 1.2 1.5 4.0 st 3 0.4 1.5 1.9 3.8 st 12 0.3 1.2 1.5 4.0 st 4 0.4 1.5 1.9 3.8 st 13 0.3 1.1 1.4 3.7 st 5 0.4 1.4 1.8 3.5 st 14 0.4 1.0 1.4 2.5 sm 6 0.4 1.4 1.8 3.5 st 15 0.4 1.0 1.4 2.5 sm 7 0.4 1.4 1.8 3.5 st 16 0.4 1.0 1.4 2.5 sm 8 0.4 1.4 1.8 3.5 st 17 0.5 0.9 1.4 1.8 sm 9 0.5 1.3 1.8 2.6 sm 18 0.5 0.8 1.3 1.6 m 10 0.5 1.3 1.8 2.6 sm 19 0.6 0.7 1.3 1.2 m 11 0.5 1.2 1.7 2.4 sm 20 0.6 0.7 1.3 1.2 m 12 0.5 1.2 1.7 2.4 sm 21 0.3 0.9 1.2 3.0 sm 13 0.4 1.2 1.6 3.0 sm 22 0.3 0.9 1.2 3.0 sm 14 0.4 1.2 1.6 3.0 sm 23 0.4 0.8 1.2 2.0 sm 15 0.5 0.9 1.4 1.8 sm 24 0.4 0.8 1.2 2.0 sm 16 0.5 0.9 1.4 1.8 sm 25 0.4 0.8 1.2 2.0 sm 17 0.7 0.7 1.4 1.0 M 26 0.4 0.8 1.2 2.0 sm 18 0.7 0.7 1.4 1.0 M 27 0.5 0.7 1.2 1.4 m 19 0.7 0.7 1.4 1.0 M 28 0.5 0.7 1.2 1.4 m 20 0.7 0.7 1.4 1.0 M 29 0.5 0.7 1.2 1.4 m 21 0.5 0.8 1.3 1.6 m 30 0.5 0.7 1.2 1.4 m 22 0.5 0.8 1.3 1.6 m 31 0.5 0.6 1.1 1.2 m 23 0.4 0.9 1.3 2.3 sm 32 0.4 0.7 1.1 1.8 sm 24 0.4 0.9 1.3 2.3 sm 33 0.4 0.7 1.1 1.8 sm 25 0.6 0.7 1.3 1.2 m 34 0.3 0.7 1.0 2.3 sm 26 0.6 0.7 1.3 1.2 m 35 0.3 0.7 1.0 2.3 sm 27 0.6 0.6 1.2 1.0 M 36 0.4 0.6 1.0 1.5 m 28 0.6 0.6 1.2 1.0 M 37 0.5 0.5 1.0 1.0 M 29 0.3 0.8 1.1 2.7 sm 38 0.5 0.5 1.0 1.0 M 30 0.3 0.8 1.1 2.7 sm 39 0.5 0.5 1.0 1.0 M 31 0.4 0.7 1.1 1.8 sm 40 0.5 0.5 1.0 1.0 M 32 0.4 0.7 1.1 1.8 sm 41 0.4 0.5 0.9 1.3 m 33 0.4 0.7 1.1 1.8 sm 42 0.4 0.5 0.9 1.3 m 34 0.4 0.7 1.1 1.8 sm 43 0.4 0.4 0.8 1.0 M 35 0.3 0.7 1.0 2.3 sm 44 0.4 0.4 0.8 1.0 M 36 0.3 0.6 0.9 2.0 sm 45 0.3 0.4 0.7 1.3 m 2010 Cytogeography of Glechoma hederacea subsp. grandis (Labiatae) in Japan 259
10 metacentric, 18 submetacentric, and 8 Table 3. Masurements of somatic metaphase subtelocentric chromosomes. The karyotype chromosomes of 2n 54 Glechoma hederacea subsp. grandis was represented by the equation: 6M 4m 18sm 8st for tetraploids. Chromosome No. Length (mm) Arm ratio Pentaploid plants had chromosomes at form metaphase which ranged from 0.7 mm to 1 0.5 1.3 1.8 2.6 sm 1.7 mm in length, and had an arm ratio between 2 0.6 1.2 1.8 2.0 sm 1.0 and 4.7 (Fig. 4B). As highlighted in Table 3 0.6 1.2 1.8 2.0 sm 2, the 45 chromosomes were divided into 3 4 0.6 1.2 1.8 2.0 sm groups: 21 metacentric, 19 submetacentric, and 5 0.6 1.2 1.8 2.0 sm 6 0.6 1.2 1.8 2.0 sm 5 subtelocentric chromosomes. The karyotype 7 0.4 1.2 1.6 3.0 sm was represented by the equation: 6M 15m 8 0.4 1.2 1.6 3.0 sm 19sm 5st for pentaploids. 9 0.4 1.2 1.6 3.0 sm Finally, hexaploid plants had 10 0.4 1.2 1.6 3.0 sm 11 0.3 1.2 1.5 4.0 st chromosomes at metaphase which ranged from 12 0.3 1.2 1.5 4.0 st 0.8 mm to 1.8 mm in length, and had an arm 13 0.6 0.9 1.5 1.5 m ratio between 1.0 and 4.0 (Fig. 4C). As 14 0.6 0.9 1.5 1.5 m 15 0.6 0.9 1.5 1.5 m shown in Table 3, the 54 chromosomes were 16 0.6 0.9 1.5 1.5 m divided into 3 groups: 32 metacentric, 20 17 0.5 0.9 1.4 1.8 sm submetacentric, and 2 subtelocentric 18 0.5 0.9 1.4 1.8 sm chromosomes. Furthermore, the karyotype 19 0.4 1.0 1.4 2.5 sm 20 0.4 1.0 1.4 2.5 sm was represented by the equation: 21 0.5 0.8 1.3 1.6 m 6M 26m 20sm 2st for hexaploids. 22 0.5 0.8 1.3 1.6 m Diploid plants for G. hederacea with 23 0.4 0.9 1.3 2.3 sm 2n 18 chromosomes have been reported 24 0.4 0.9 1.3 2.3 sm 25 0.5 0.7 1.2 1.4 m (Sugiura 1940 as Nepeta Glechoma Benth., 26 0.5 0.7 1.2 1.4 m Suzuka 1950, Suzuka and Koriba 1952, Sorsa 27 0.5 0.7 1.2 1.4 m 1963, Laane 1971, Gill 1979, 1981). However, 28 0.5 0.7 1.2 1.4 m the present study showed that diploid plants do 29 0.5 0.7 1.2 1.4 m 30 0.5 0.7 1.2 1.4 m not occur in Japan for G. hederacea subsp. 31 0.5 0.7 1.2 1.4 m grandis. Polyploid variations of 2n 18, 36, 45, 32 0.5 0.7 1.2 1.4 m 54 found in G. hederacea suggests that the 33 0.6 0.6 1.2 1.0 M plant with 2n 54 chromosomes is an 34 0.6 0.6 1.2 1.0 M 35 0.5 0.7 1.2 1.4 m amphiploid plant originated by hybridization 36 0.5 0.7 1.2 1.4 m between diploid (2n 18) and tetraploid 37 0.4 0.8 1.2 2.0 sm (2n 36) plants. Within the 3 chromosomal 38 0.4 0.8 1.2 2.0 sm forms of G. hederacea subsp. grandis, 39 0.4 0.8 1.2 2.0 sm 40 0.4 0.8 1.2 2.0 sm pentaploids were considered to be hybrids 41 0.5 0.6 1.1 1.2 m between tetraploid and hexaploid plants 42 0.5 0.6 1.1 1.2 m (Iwatsubo et al. 2004). The present study 43 0.5 0.6 1.1 1.2 m 44 0.5 0.6 1.1 1.2 m shows that the karyotype formula 45 0.5 0.6 1.1 1.2 m (6M 15m 19sm 5st) for pentaploid plants 46 0.5 0.6 1.1 1.2 m corresponds to half the chromosomal set 47 0.5 0.5 1.0 1.0 M 48 0.5 0.5 1.0 1.0 M (3M 2m 9sm 4st) of tetraploid and half the 49 0.4 0.5 0.9 1.3 m chromosomal set (3M 13m 10sm 1st) of 50 0.4 0.5 0.9 1.3 m hexaploid plants. Moreover, pentaploid plants 51 0.3 0.5 0.8 1.7 m exist only in regions where tetraploid and 52 0.3 0.5 0.8 1.7 m hexaploid plant distributions overlap. Our 53 0.4 0.4 0.8 1.0 M 54 0.4 0.4 0.8 1.0 M results on both the distribution and karyotype 260 N. Miura and Y. Iwatsubo Cytologia 75(3) for the 3 chromosomal forms of G. hederacea subsp. grandis in Japan also show that pentaploids are hybrid plants between tetraploid and hexaploid plants.
Acknowledgements We are grateful to Ms. M. Ando, Ms. M. Chikamori, Mr. R. Enomoto, Ms. A. Ishiyama, Mr. K. Ito, Mr. G. Kodate, Ms. M. Matsuda, Mr. H. Nakamura, Ms. M. Nakamura, Ms. K. Ogino, Mr. S. Ohta, Mr. H. Saito, Mr. K. Sasamura, Dr. K. Sato, Mr. T. Sawanomukai, Ms. Y. Souma, Mr. M. Sugimoto, Mr. T. Takanashi, Ms. M. Takashima, Mr. K. Tanaka, Mr. H. Ueda, Mr. K. Yamamoto, Mr. T. Yamazaki and Mr. M. Yauchi for providing us with plant materials. We would also like to thank Mr. M. Sugimoto and Dr. N. Naruhashi for their valuable help and useful comments. This work was supported in part by a Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science (no. 20011439) granted to N. Miura.
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