Cytogeography of Taraxacum venustum and T. shikotanense (Asteraceae) in Japan

Abstract

We report on polyploidy, karyotypes, and cytogeographical distribution of two species of native agamospermous Taraxacum in Japan. Taraxacum venustum had two polyploid numbers of 2n=3x=24 and 2n=4x=32. Four karyotypes were found in tetraploid plants. In T. shikotanense, there were seven chromosome numbers: 2n=47 (hypohexaploid), 48 (hexaploid), 56 (heptaploid), 64 (octoploid), 72 (nonaploid), 80 (decaploid), and 88 (undecaploid). Our study reveals that some races in both T. venustum and T. shikotanense can be differentiated according to their polyploidy and karyotypes.

The genus Taraxacum (Asteraceae) comprises approximately 15 species in Japan (Morita 2017). This is a monobasic genus with x=8 (Darlington and Wylie 1955). Diploid plants are sexual, whereas the polyploid plants are mostly obligatory but sometimes facultatively agamospermous (Richards 1970, Grant 1981).

T. venustum H. Koidz. is a perennial herb that is distributed in Hokkaido and north to central Honshu in Japan, as well as Sakhalin in the Russian Federation (Morita 1995, 2017). The chromosome number of T. venustum has been reported to be 2n=24 (Okabe 1934, 1951, Matsuura and Sutô 1935, Takemoto 1961, 1970, Yamaguchi 1976, Akhter et al. 1993), 32 (Nishikawa 1984, Akhter et al. 1993), and 40 (Akhter et al. 1993) as T. hondoense. These earlier reports found that T. venustum has intraspecific polyploidy (3x, 4x, and 5x).

Akhter et al. (1993) surveyed chromosome numbers and allozyme variations of T. venustum (as T. hondoense Nakai ex H. Koidz.) distributed in northern Honshu, Japan. The study identified a total of 21 clones using three enzyme systems, revealing a considerable amount of clonal diversity both within and among polyploid populations. However, they did not investigate T. venustum distributed in Hokkaido.

T. shikotanense Kitam., the congener, is distributed in eastern coastal areas of Hokkaido (Morita 1995, 2017). The chromosome number for T. shikotanense has been reported to be 2n=64 chromosomes (Okabe 1951, Takemoto 1956, 1961, Yamaguchi 1976).

In the present study, we elucidated the cytogeographical structure of T. venustum and T. shikotanense by covering their entire distribution areas in Japan, and present an extensive study of its karyotypes.

Materials and methods

A total of 735 plants of T. venustum, collected from 111 localities in Japan (Table 1), 464 plants of T. shikotanense from 27 localities in Hokkaido Pref. (Table 2), were examined. The plants were grown in plastic pots at the experimental garden of the University of Toyama.

Table 1. Chromosome number, collection locality and number of individuals examined of T. venustum.

Collection locality	Chromosome number (2n)		Total
	24	32
Hokkaido Pref.
Akancho-Nishiakan, Kushiro City		3	3
Atoekamura, Kushiro-cho, Kushiro-gun		3	3
Bansei, Taiki-cho, Hiroo-gun	1	4	5
Chobushi, Toyokoro-cho, Nakagawa-gun		2(1, type I)*	2
Hamakoshimizu, Koshimizu-cho, Shari-gun		13	13
Higashiohnuma, Nanae-cho, Kameda-gun		13(4, type II)	13
Kamiikusagawa, Nanae-cho, Kameda-gun		3(1, type II)	3
Koitoi, Shiranuka-cho, Shiranuka-gun		3	3
Maruyama, Tomakomai City		17	17
Miharashicho, Hakodate City		12	12
Minamioka, Kitami City	7	5	12
Mitsuura, Kushiro City		1	1
Nishikimachi, Makubetsu-cho, Nakagawa-gun		6	6
Nozuka, Hiroo-cho, Hiroo-gun		16(1, type I)	16
Ohtsu, Toyokoro-cho, Nakagawa-gun		5	5
Onbetsucho-Nakanobetsu, Kushiro City		5	5
Rakuyo, Nemuro City		1	1
Rubeshibetsu, Hiroo-cho, Hiroo-gun		3	3
Seika, Taiki-cho, Hiroo-gun		6	6
Shincho, Chitose City		1	1
Shoya, Erimo-cho, Horoizumi-gun		1	1
Tokorocho-Higashihama, Kitami City	1		1
Toyo, Erimo-cho, Horoizumi-gun		7	7
Tsuruoka, Kushiro City		8(1, type I)	8
Uenae, Tomakomai City		2	2
Yoro, Urahoro-cho, Tokachi-gun	2		2
Yudo, Toyokoro-cho, Nakagawa-gun	14(1)	14(2. type I)	28
Yunoshima, Teshikaga-cho, Kawakami-gun		3(1, type II)	3
Aomori Pref.
Ikarigaseki, Hirakawa City	3		3
Ishikawa, Hirosaki City		3	3
Nijikai, Ohwani-machi, Minamitsugaru-gun		6	6
Iwate Pref.
Kawauchi, Miyako City		8	8
Moichi, Miyako City	5		5
Akita Pref.
Kanezawa, Misato-cho, Senboku-gun	9		9
Noshiromachi, Noshiro City	1		1
Ono, Yuzawa City	6		6
Tazawako-sotsuda, Senboku City	2		2
Miyagi Pref.
Hanayama-honsawa, Kurihara City	5		5
Hanayama-kusakisawa, Kurihara City	2(2)	5	7
Hasamacho-Sanuma, Tome City	5		5
Hatsubara, Matsushima-machi, Miyagi-gun	4(1)		4
Ichihasama-kosakamoto, Kurihara City	2		2
Iwadeyama-iketsuki, Ohsaki City	10(1)		10
Matsushima, Matsushima-machi, Miyagi-gun	8		8
Miya, Zao-machi, Katta-gun	6		6
Motoyoshicho-Magomemachi, Kesennuma City	4	1	5
Nakamura, Ohsato-cho, Kurokawa-gun	9		9
Nishihunabasama, Shibata-machi, Shibata-gun	2		2
Obara, Shiroishi City	1(1)	1	2
Shikama, Shikama-cho, Kami-gun	5		5
Towacho-Yonekawa, Tome City	3		3
Yamagata Pref.
Iritazawa, Yonezawa City	11	2	13
Matsubara, Iide-machi, Nishiokitama-gun	10		10
Nakada, Kaneyama-machi, Mogami-gun	5		5
Oguni-kosakamachi, Oguni-machi, Nishiokitama-gun	1		1
Fukushima Pref.
Ebana, Sukagawa City	4		4
Higashiniidono, Nihonmatsu City	5	3	8
Ichinaka-sanbanko, Aizubange-machi, Kawanuma-gun	3		3
Kawamaemachi-Ojiroi, Iwaki City	6	2	8
Kitayama, Kitashiobara-mura, Yama-gun	17		17
Makinouchi, Ten-ei-mura, Iwase-gun	6		6
Mutsuai, Nishiaizu-machi, Yama-gun	9	2	11
Nishigodo, Hanawa-machi, Higashishirakawa-gun	5		5
Ogawamachi-Kamiogawa, Iwaki City	10(1)*	4(2, type IV)	14
Ogawamachi-Shioda, Iwaki City	6	2	8
Ohshio, Kitashiobara-mura, Yama-gun	3	1	4
Ohta, Nihonmatsu City	4		4
Ohtsunagi, Kawamata-machi, Date-gun	10		10
Ojima, Kawamata-machi, Date-gun	9(3)		9
Sakamoto, Aizubange-machi, Kawanuma-gun	10		10
Tsuruzawa, Kawamata-machi, Date-gun	4		4
Watarase, Samegawa-mura, Higashishirakawa-gun	19		19
Watarase-Aono, Samegawa-mura, Higashishirakawa-gun		5(2, type III)	5
Yanaizu, Yanaizu-machi, Kawanuma-gun	9	1	10
Niigata Pref.
Hirabori, Aga-machi, Higashikanbara-gun		11	11
Ichinotsubo, Izumozaki-machi, Santo-gun	4		4
Itsukamachi, Minamiuonuma City		1	1
Kumawatari, Aga-machi, Higashikanbara-gun		1	1
Nishiyamacho-Sakata, Kashiwazaki City	13		13
Niwazuki, Sanjo City	2	5	7
Ohmaki, Aga-machi, Higashikanbara-gun	2		2
Shimazaki, Nagaoka City	1	2	3
Yatsuda, Aga-machi, Higashikanbara-gun	5		5
Yazawa, Aga-machi, Higashikanbara-gun	10		10
Gunma Pref.
Misatomachi-Nishiakiya, Takasaki City	7		7
Toyama Pref.
Ashikuraji, Tateyama-machi, Nakaniikawa-gun	27		27
Fukumitsu, Nanto City	5(1)		5
Fukuokamachi-Goi, Takaoka City	3(1)		3
Fukuokamachi-Shimomukuta, Takaoka City	5(1)		5
Fushikikofu, Takaoka City	5		5
Futomi, Nanto City	3		3
Gofuku, Toyama City	12(1)		12
Gotani, Tonami City	15		15
Inami, Nanto City	3		3
Kasuga, Nyuzen-machi, Shimoniikawa-gun	3(1)		3
Kawanishi, Nanto City	15		15
Kitadai, Toyama City	10		10
Kitayashiro, Himi City	2		2
Minowa, Oyabe City	7		7
Ohzakai, Himi City	1		1
Sannokuma, Toyama City	7		7
Sasagawa, Asahi-machi, Shimoniikawa-gun	5		5
Shiraiwa, Tateyama-machi, Nakaniikawa-gun	11		11
Shogawamachi-Kanaya, Tonami City	10		10
Tagawa, Oyabe City	4		4
Takabatake, Asahi-machi, Shimoniikawa-gun	13		13
Tsubono, Tonami City	5		5
Wariyama, Toyama City	7(1)		7
Yatsuomachi-Nakajinzu, Toyama City	2		2
Gifu Pref.
Kamitakaracho-Nezumimochi, Takayama City	1		1
Ishikawa Pref.
Tomita, Tsubata-machi, Kahoku-gun	4		4
Total	512	223	735

*Numbers in parentheses are the individuals used karyotype analysis.

　

Table 2. Chromosome number, collection locality and number of individuals examined of T. shikotanense.

Collection locality	Chromosome number (2n)							Total
	47	48	56	64	72	80	88
Hokkaido Pref.
Bansei, Taiki-cho, Hiroo-gun	3	9		16				28
Bihoro, Hiroo-cho, Hiroo-gun		2	11(1)*					13
Bitatanunke, Hiroo-cho, Hiroo-gun							10(1)	10
Chobushi, Toyokoro-cho, Nakagawa-gun			1	16				17
Erimomisaki, Erimo-cho, Horoizumi-gun					8(1)	18		26
Funbe, Hiroo-cho, Hiroo-gun				20				20
Hamataiki, Taiki-cho, Hiroo-gun		47						47
Katsurakoi, Kushiro City				5	6			11
Koitoi, Shiranuka-cho, Shiranuka-gun				4				4
Masuura, Kushiro City				2	42(1)			44
Meguro, Erimo-cho, Horoizumi-gun				4		6		10
Mitsuura, Kushiro City				1	3			4
Moyori, Hiroo-cho, Hiroo-gun				17				17
Niino, Kushiro City				5	1			6
Nozuka, Hiroo-cho, Hiroo-gun		55(1)						55
Onbetsucho-Nakaonbetsu, Kushiro City			5	15				20
Ohtsumotomachi, Toyokoro-cho, Nakagawa-gun				9				9
Oshirabetsu, Hiroo-cho, Hiroo-gun				10				10
Otanoshike-minami, Kushiro City				12				12
Rubeshibetsu, Hiroo-cho, Hiroo-gun						2	3	5
Shinfujicho, Kushiro City					5			5
Shoya, Erimo-cho, Horoizumi-gun			1	20(1)				21
Taniiso, Hiroo-cho, Hiroo-gun							3	3
Tokachibuto, Urahoro-cho, Tokachi-gun				10				10
Toyo, Erimo-cho, Horoizumi-gun				3		15(1)		18
Utabetsu, Erimo-cho, Horoizumi-gun						2		2
Yudo, Toyokoro-cho, Nakagawa-gun		2	13	21	1			37
Total	3	115	31	190	66	43	16	464

*Numbers in parentheses are the individuals used karyotype analysis.

Newly formed root tips collected from the potted plants were pretreated in 2.1 mM 8-hydroxyquinoline at 25°C for 1 to 1.5 h and kept at ca. 5°C for 15 h. The root tips were fixed in a mixture of glacial acetic acid and absolute ethyl alcohol (1 : 3) at 25°C for 1 h, macerated in 1 M hydrochloric acid at 60°C for 10 min, and then washed in tap water. Root tip meristems were stained using 1.5% lacto-propionic orcein on a glass slide, and a conventional squash technique was applied in the preparation. Their chromosomes were examined in fully spread metaphase chromosomes from meristematic cells. Chromosome forms were described according to the nomenclature of Tanaka (1977). The chromosome forms in the present study were defined as follows: M (median s.s.)=arm ratio is 1.0, median=arm ratios above 1.1 and below 1.7, submedian=arm ratios above 1.8 and below 3.0. The cells used for karyotype analysis were at least 10 per individual.

For meiotic chromosome pairing, young flower buds were prepared in Newcomer’s fixative at 17°C for 3 h. The buds and anthers were macerated, then stained and squashed using the same procedure described above for root tips. The chromosome pairing at metaphase I was studied in pollen mother cells (PMCs) of 3x, 4x of T. venustum and 9x T. shikotanense plants.

Results

Chromosome numbers

T. venustum had two cytotypes: triploid plants with 2n=24, tetraploid plants with 2n=32 (Table 1, Fig. 1). As shown in Table 1, 512 plants were triploid (69.7%) and 223 plants were tetraploid (30.3%), respectively. There were no pentaploid plants with 2n=5x=40 chromosomes, although such plants have been identified by Akhter et al. (1993) in Nagano Pref. and Niigata Pref. in central Honshu.

Fig. 1. Photographs of somatic metaphase chromosomes of two species of Taraxacum. A, B: T. venustum; C–I: T. shikotanense. A: 2n=24; B: 2n=32; C: 2n=47; D: 2n=48; E: 2n=56; F: 2n=64; G: 2n=72; H: 2n=80; I: 2n=88. Scale bars=5 µm.

T. shikotanense had seven cytotypes: hypohexaploid plants with 2n=47 (6x−1), hexaploid plants with 2n=48 (6x), heptaploid plants with 2n=56 (7x), octoploid plants with 2n=64 (8x), nonaploid plants with 2n=72 (9x), decaploid plants with 2n=80 (10x), and undecaploids with 2n=88 (11x) (Table 2, Fig. 1). The 2n=64 (8x) is agreement with previous reports (Okabe 1951, Takemoto 1956, 1961, Yamaguchi 1976). This species has been known as an octoploid agamospermous dandelion. However, the results of the present study show that T. shikotanense is composed of hypohexaploid to undecaploid. Out of 464 plants examined, 115 (24.8%) were hexaploids, 31 (6.7%) were heptaploids, 190 (40.9%) were octoploids, 66 (14.2%) were nonaploids, 43 (9.3%) were decaploids, 16 (3.4%) were undecaploids, and three plants were hypohexaploids (0.6%).

Cytogeographic distribution

In T. venustum, triploid and tetraploid plants were found in a wide range from Hokkaido to Honshu (Fig. 2). Tetraploid plants, however, were not found in the southernmost distribution area: Gunma, Toyama, Gifu, and Ishikawa Prefs. in Honshu (Fig. 2B).

Fig. 2. Distribution of T. venustum. A: 2n=24; B: 2n=32. Detailed localities are provided in Table 1. Scale bar=100 km.

In T. shikotanense, octoploid (2n=64) plants, found in 18 sites, were most widely distributed throughout the distribution areas of this species in Hokkaido (Fig. 3). Plural different polyploids were often found sympatrically (Table 2).

Fig. 3. Distribution of T. shikotanense. A: 2n=47; B: 2n=48; C: 2n=56; D: 2n=64; E: 2n=72; F: 2n=80; G: 2n=88. Detailed localities are provided in Table 2. Scale bar=100 km.

Karyotype analysis

Triploid (2n=24) of T. venustum (Table S1, Fig. 4A)

Fig. 4. Karyotypes of somatic metaphase chromosomes of T. venustum. A: 2n=24 plant collected in ; B: 2n=32 (type I) plant collected in in Yudo, Toyokoro-cho, Nakagawa-gun, Hokkaido Pref.; C: 2n=32 (type II) plant collected in Higashiohnuma, Nanae-cho, Kameda-gun, Hokkaido Pref.; D: 2n=32 (type III) plant collected in Watarase-Aono, Samegawa-mura, Higashishirakawa-gun, Fukushima Pref.; E: 2n=32 (type IV) plant collected in Ogawamachi-Kamiogawa, Iwaki City, Fukushima Pref. Arrows indicate chromosome arms with secondary constrictions. Scale bar=5 µm.

All 16 individuals collected from 13 localities had the same karyotypes in current study (Table 1). Chromosomes at metaphase ranged from 1.0 to 3.8 µm in length and 1.1 to 2.3 in arm ratio. The 24 chromosomes were divided into two groups: 22 metacentric chromosomes and two submetacentric chromosomes (Table S1). In the chromosome complement, one metacentric chromosome had a secondary constriction of its short arm, and five metacentric chromosomes had a secondary constriction in each of their long arms. The total length of the complement was 58.5 µm and the longest chromosome to the shortest chromosome ratio was 3.8. The chromosome complement of this plant was formulated as 2n=24=16m+1m^cs+5m_cs+2sm.

Consistent with our present findings, Takemoto (1961) reported that 3x T. venustum (as T. hondoense) has six chromosomes with a secondary constriction in the complement.

Tetraploid (2n=32) of T. venustum (Tables S2–S5, Fig. 4B–E)

Four karyotypes (type I, II, III, and IV) were found in tetraploid plants. Comparison of each karyotype is summarized in the Table 3.

Table 3. Comparison of four different karyotypes of 4x T. venustum found in this study.

Type	No. of chromosomes				Karyotype formulae	No. of satellited chromosomes			Range of arm ratio	Range of length (µm)	Total length (µm)	Longest chromosome length/shortest chromosome length
	M	m	sm	Total		m	sm	Total
I	1	27	4	32	1M+23m+1mcs+3mcs+4sm	4	0	4	1.0–2.1	1.5–3.8	74.9	2.5
II	0	29	3	32	24m+5mcs+3sm	5	0	5	1.1–2.1	1.6–3.8	81.0	2.4
III	0	30	2	32	27m+3mcs+1sm+1smcs	3	1	4	1.1–1.9	1.4–3.8	73.0	2.7
IV	1	28	3	32	1M+23m+1mcs+4mcs+3sm	5	0	5	1.0–2.2	1.1–3.4	66.7	3.1

In 4x type I karyotype, chromosomes at metaphase ranged from 1.5 to 3.8 µm in length and 1.0 to 2.1 in arm ratio. The 32 chromosomes were divided into two groups: 28 metacentric chromosomes and four submetacentric chromosomes (Table S2). In the chromosome complement, one metacentric chromosome had a secondary constriction of its short arm, and three metacentric chromosomes had a secondary constriction in each of their long arms. The total length of the complement was 74.9 µm and the longest chromosome to the shortest chromosome ratio was 2.5. The chromosome complement of this plant was formulated as 2n=32=1M+23m+1m^cs+3m_cs+4sm.

In 4x type II karyotype, chromosomes at metaphase ranged from 1.6 to 3.8 µm in length and 1.1 to 2.1 in arm ratio. The 32 chromosomes were divided into two groups: 29 metacentric chromosomes and three submetacentric chromosomes (Table S3). In the chromosome complement, five metacentric chromosomes had a secondary constriction in each of their long arms. The total length of the complement was 81.0 µm and the longest chromosome to the shortest chromosome ratio was 2.4. The chromosome complement of this plant was formulated as 2n=32=24m+5m_cs+3sm.

In 4x type III karyotype, chromosomes at metaphase ranged from 1.4 to 3.8 µm in length and 1.1 to 1.9 in arm ratio. The 32 chromosomes were divided into two groups: 30 metacentric chromosomes and two submetacentric chromosomes (Table S4). In the chromosome complement, three metacentric chromosomes and one submetacentric chromosome had a secondary constriction in each of their long arms. The total length of the complement was 73.0 µm and the longest chromosome to the shortest chromosome ratio was 2.7. The chromosome complement of this plant was formulated as 2n=32=27m+3m_cs+1sm+1sm_cs.

In 4x type IV, chromosomes at metaphase ranged from 1.1 to 3.4 µm in length and 1.0 to 2.2 in arm ratio. The 32 chromosomes were divided into two groups: 29 metacentric chromosomes and three submetacentric chromosomes (Table S5). In the chromosome complement, one metacentric chromosome had a secondary constriction of its short arm, and four metacentric chromosomes had a secondary constriction in each of their long arms. The total length of the complement was 66.7 µm and the longest chromosome to the shortest chromosome ratio was 3.1. The chromosome complement of this plant was formulated as 2n=32=1M+23m+1m^cs+4m_cs+3sm.

Hexaploid (2n=48) of T. shikotanense (Table S6, Fig. 5)

Fig. 5. Karyotypes of somatic metaphase chromosomes of 6x T. shikotanense collected in Nozuka, Hiroo-cho, Hiroo-gun, Hokkaido Pref. Arrows indicate chromosome arms with secondary constrictions. Scale bar=5 µm.

Chromosomes at metaphase ranged from 1.6 to 3.6 µm in length and 1.0 to 2.0 in arm ratio. The 48 chromosomes were divided into two groups: 46 metacentric chromosomes and two submetacentric chromosomes (Table S6). In the chromosome complement, one metacentric chromosome had a secondary constriction of its short arm, and five metacentric chromosomes had a secondary constriction in each of their long arms. The total length of the complement was 113.1 µm and the longest chromosome to the shortest chromosome ratio was 2.3. The chromosome complement of this plant was formulated as 2n=48=1M+39m+1m^cs+5m_cs+2sm.

Heptaploid (2n=56) of T. shikotanense (Table S7, Fig. 6)

Fig. 6. Karyotypes of somatic metaphase chromosomes of 7x T. shikotanense collected in Bihoro, Hiroo-cho, Hiroo-gun, Hokkaido Pref. Arrows indicate chromosome arms with secondary constrictions. Scale bar=5 µm.

Chromosomes at metaphase ranged from 1.5 to 3.1 µm in length and 1.0 to 1.7 in arm ratio. The chromosome complement of this plant was composed only of metacentric chromosomes (Table S7). In the chromosome complement, one metacentric chromosome had a secondary constriction of its short arm, and four metacentric chromosomes had a secondary constriction in each of their long arms. The total length of the complement was 104.2 µm and the longest chromosome to the shortest chromosome ratio was 2.1. The chromosome complement of this plant was formulated as 2n=56=7M+44m+1m^cs+4m_cs.

Octoploid (2n=64) of T. shikotanense (Table S8, Fig. 7)

Fig. 7. Karyotypes of somatic metaphase chromosomes of 8x T. shikotanense collected in Shoya, Erimo-cho, Horoizumi-gun, Hokkaido Pref. Arrows indicate chromosome arms with secondary constrictions. Scale bar=5 µm.

Chromosomes at metaphase ranged from 1.3 to 3.7 µm in length and 1.0 to 2.0 in arm ratio. The 64 chromosomes were divided into two groups: 62 metacentric chromosomes and two submetacentric chromosomes (Table S8). In the chromosome complement, one metacentric chromosome had a secondary constriction of its short arm, and three metacentric chromosomes and one submetacentric chromosome had a secondary constriction in each of their long arms. The total length of the complement was 138.7 µm and the longest chromosome to the shortest chromosome ratio was 2.8. The chromosome complement of this plant was formulated as 2n=64=3M+55m+1m^cs+3m_cs+1sm+1sm_cs.

Nonaploid (2n=72) of T. shikotanense (Table S9, Fig. 8)

Fig. 8. Karyotypes of somatic metaphase chromosomes of 9x T. shikotanense collected in Erimomisaki, Erimo-cho, Horoizumi-gun, Hokkaido Pref. Arrows indicate chromosome arms with secondary constrictions. Scale bar=5 µm.

Chromosomes at metaphase ranged from 0.9 to 3.3 µm in length and 1.0 to 1.9 in arm ratio. The 72 chromosomes were divided into two groups: 68 metacentric chromosomes and four submetacentric chromosomes (Table S9). In the chromosome complement, one metacentric chromosome had a secondary constriction of its short arm, and four metacentric chromosomes had a secondary constriction in each of their long arms. The total length of the complement was 161.4 µm and the longest chromosome to the shortest chromosome ratio was 3.7. The chromosome complement of this plant was formulated as 2n=72=2M+60m+2m^cs+4m_cs+4sm.

Decaploid (2n=80) of T. shikotanense (Table S10, Fig. 9)

Fig. 9. Karyotypes of somatic metaphase chromosomes of 10x T. shikotanense collected in Toyo, Erimo-cho, Horoizumi-gun, Hokkaido Pref. Arrows indicate chromosome arms with secondary constrictions. Scale bar=5 µm.

Chromosomes at metaphase ranged from 1.5 to 3.4 µm in length and 1.0 to 2.7 in arm ratio. The 80 chromosomes were divided into two groups: 75 metacentric chromosomes and five submetacentric chromosomes (Table S10). In the chromosome complement, one metacentric chromosome had a secondary constriction of its short arm, and three metacentric chromosomes had a secondary constriction in each of their long arms. The total length of the complement was 196.4 µm and the longest chromosome to the shortest chromosome ratio was 2.3. The chromosome complement of this plant was formulated as 2n=80=1M+70m+1m^cs+3m_cs+5sm.

Undecaploid (2n=88) of T. shikotanense (Table S11, Fig. 10)

Fig. 10. Karyotypes of somatic metaphase chromosomes of 11x T. shikotanense collected in Bitatanunke, Hiroo-cho, Hiroo-gun, Hokkaido Pref. Arrows indicate chromosome arms with secondary constrictions. Scale bar=5 µm.

Chromosomes at metaphase ranged from 1.5 to 3.2 µm in length and 1.0 to 3.0 in arm ratio. The 88 chromosomes were divided into two groups: 79 metacentric chromosomes and nine submetacentric chromosomes (Table S11). In the chromosome complement, one metacentric chromosome had a secondary constriction of its short arm, three metacentric chromosomes had a secondary constriction in each of their long arms, and one submetacentric chromosome had a secondary constriction in its long arm. The total length of the complement was 194.7 µm and the longest chromosome to the shortest chromosome ratio was 2.1. The chromosome complement of this plant was formulated as 2n=88=3M+72m+1m^cs+3m_cs+8sm+1sm_cs.

Meiosis of PMCs

Meiotic chromosomes at metaphase I were examined in PMCs of triploid T. venustum, tetraploid T. venustum, and nonaploid T. shikotanense plants (Tables 4–6, Fig. 11). In all three plants examined, PMCs showed abnormal microsporogenesis.

Table 4. Number of pollen mother cells with different chromosome configurations at metaphase I of 3x T. venustum collectec in Kasuga, Nyuzen-machi, Shimoniikawa-gun, Toyama Pref.

Configuration	No. of PMCs
2II+20I	22
1II+22I	40
24I	148
Total	210

Table 5. Number of pollen mother cells with different chromosome configurations at metaphase I of 4x T. venustum collected in Nozuka, Hiroo-cho, Hiroo-gun, Hokkaido Pref.

Configuration	No. of PMCs
5IV+3III+3I	1
4IV+4III+4I	4
2IV+6III+6I	4
1IV+7III+7I	1
8III+8I	14
7III+1II+9I	7
6III+2II+10I	5
5III+3II+11I	5
4III+4II+12I	4
3III+5II+13I	4
2III+6II+14I	4
1III+7II+15I	6
8II+16I	3
Total	62

Table 6. Number of pollen mother cells with different chromosome configurations at metaphase I of 9x T. shikotanense collected in Masuura, Kushiro City, Hokkaido Pref.

Configuration	No. of PMCs
23II+26I	1
22II+28I	1
21II+30I	1
20II+32I	1
19II+34I	1
18II+36I	1
17II+38I	1
16II+40I	1
15II+42I	1
14II+44I	1
13II+46I	1
12II+48I	1
11II+50I	1
10II+52I	1
8II+56I	5
7II+58I	2
6II+60I	3
4II+64I	3
3II+66I	3
2II+68I	2
1II+70I	8
72I	9
Total	49

Fig. 11. Photographs of first meiotic metaphase in PMCs of Taraxacum species. A: 2n=24 of T. venustum collected in Kasuga, Nyuzen-machi, Shimoniikawa-gun, Toyama Pref.; B: 2n=32 of T. venustum collected in Nozuka, Hiroo-cho, Hiroo-gun, Hokkaido Pref.; C: 2n=72 of T. shikotanense collected in Masuura, Kushiro City, Hokkaido Pref. Scale bar=5 µm.

In triploid plant of T. venustum, chromosome pairing at metaphase I was examined in 210 PMCs (Table 4, Fig. 11A). There were various numbers of univalents and bivalents, ranging from 20–24 and 0–2, respectively (Table 4). The most frequent form of chromosome association was 24_I (70.5%), followed by 1_II+22_I (19.0%) and 2_II+20_I (10.5%). The mean chromosome pairing per cell was 0.4_II+23.2_I.

In tetraploid plant of T. venustum, chromosome pairing at metaphase I was examined in 62 PMCs (Table 5, Fig. 11B). There were various numbers of univalents, bivalents, trivalents, and tetravalents, ranging from 3–16, 0–8, 0–8, and 0–5, respectively (Table 5). The most frequent form of chromosome association was 8_III+8_I(22.6%), followed by 7_III+1_II+9_I(11.3%), 1_III+7_II+15_I(9.7%), 6_III+2_II+10_I(8.1%), 5_III+3_II+11_I(8.1%), 4_IV+4_III+4_I(6.5%), 2_IV+6_III+6_I(6.5%), 4_III+4_II+12_I(6.5%), 3_III+5_II+13_I(6.5%), 2_III+6_II+14_I(6.5%), 8_II+16_I(4.8%), 5_IV+3_III+3_I(1.6%), and 1_IV+7_III+7_I(1.6%). The mean chromosome pairing per cell was 0.5_IV+5.0_III+2.5_II+10.1_I.

In nonaploid plant of T. shikotanense, chromosome pairing at metaphase I was examined in 49 PMCs (Table 6, Fig. 11C). There were various numbers of univalents and bivalents, ranging from 26–72 and 0–23, respectively (Table 6). The most frequent form of chromosome association was 72I (18.4%), followed by 1_II+70_I(16.3%), 8_II+56_I(10.2%), 6_II+60_I(6.1%), 4_II+64_I(6.1%), 3_II+66_I(6.1%), 7_II+58_I(4.1%), 2_II+68_I(4.1%), 23_II+26_I(2.0%), 22_II+28_I(2.0%), 21_II+30_I(2.0%), 20_II+32_I(2.0%), 19_II+34_I(2.0%), 18_II+36_I(2.0%), 17_II+38_I(2.0%), 16_II+40_I(2.0%), 15_II+42_I(2.0%), 14_II+44_I(2.0%), 13_II+46_I(2.0%), 12_II+48_I(2.0%), 11_II+50_I(2.0%), and 10_II+52_I(2.0%). The mean chromosome pairing per cell was 6.9_II+58.3_I.

Discussion

The smallest two chromosomes were found to be extremely short compared to the other chromosomes in the 3x T. venustum complement (Table S1). This proves clearly that 3x T. venustum is an allopolyploid plant. No variation was detected in the triploid karyotype, suggesting that individuals with the same karyotype are widely distributed by agamospermy in Japan.

The present study suggested that chromosome inversion or translocation plays an important role in the diversification of 4x T. venustum in Japan. In tetraploid T. venustum (2n=32), four strains distinguished by their karyotypes were found. Richards (1989) demonstrated that remarkably high levels of chromosome breakage and somatic recombination occur within some Taraxacum families, and that marked variation for this trait apparently occurs between lines, even within an agamospecies. Karyotypic variation found in this study is consistent with the obtained by Richards (1989).

On the other hand, the present study showed that polyploidization performs in the diversification of T. shikotanense. Polyploidy of T. shikotanense ranged from hypohexaploid to undecaploid. Undecaploid T. shikotanense (2n=88) was identified for the first time in this study and has the highest polyploidy in the genus Taraxacum.

Meiotic chromosomes of 3x T. venustum, 4x T. venustum, and 9x T. shikotanense showed a high frequency of univalents and bivalents, while there were only a few trivalents. Chromosome pairing also indicated that 3x T. venustum, 4x T. venustum, and 9x T. shikotanense were allopolyploid.

Acknowledgments

The authors acknowledge the support of Dr. N. Miura, Mr. K. Sasamura, Dr. S. Serizawa, and Dr. M. Watanabe for collecting materials.

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