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Cytology of Micranthes fusca (Saxifragaceae) and Its Related Species

Tomoko Fukudaa,*, Andrei Loguntsevb, Igor BoBrovb, Maxim antipinb, Aleksandr taranc, Hideki takahashid

and Hiroshi ikedae

aDepartment of Botany, National Museum of Nature and Science, 4-1-1, Amakubo, Tsukuba, 305-0005 JAPAN;

bState Nature Reserve “Kurilsky”, Yuzhno-Kurilsky, Kunashiri Island;cSakhalin Botanical Garden, Gorky st., Yuzhno-Sakhalinsk, Sakhalin, RUSSIA;

dHokkaido University Museum, Kita 10, Nishi 8, Kita-ku, Sapporo, Hokkaido, 060-0810 JAPAN;e The University Museum, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 111-0033 JAPAN

*Corresponding author: fukuda@kahaku.go.jp

(Accepted on November 2, 2013)

Chromosome numbers of Micranthes fusca (Maxim.) S. Akiyama & H. Ohba and its related species were determined. The chromosome number for M. fusca was mostly 2n = 30, as in the previous reports. In addition, we found 2n = 45 among population from Kunashiri Island of the Kuril Islands. The chromosome number of Saxifraga purpurascens Kom., 2n = 24, is the first report for this species. The chromosome number 2n = 28 for M. japonica (Boissieu) S. Akiyama & H. Ohba agrees with the previous report.

Key words: Chromosome number, karyotype, Micranthes fusca, Micranthes japonica, Saxifragaceae, Saxifraga purpurascens.

J. Jpn. Bot. 89: 111–117 (2014)

Micranthes, including 67 species, is one of the largest genera of Saxifragaceae (Mort and Soltis 1999). Micranthes was recently divided as an independent genus from Saxifraga (Brouillet and Gornall 2007, Akiyama et al. 2012) based on phylogenetic analyses (e.g., Soltis et al. 1993, 2001) and also on morphological characteristics; such as leafless flowering stems, ovules with a single integument except in some species, reticulate pollen exine sculpturing and longitudinally ribbed seeds (e.g., Ferguson and Webb 1970, Brouillet and Gornall 2007). Species of the Micranthes are widely distributed mainly in the Northern Hemisphere, including several species in Japan: M. fusca (Maxim.) S. Akiyama & H. Ohba, M. japonica (Boissieu) S.

Akiyama & H. Ohba, M. nelsoniana (D. Don) Small var. reniformis (Ohwi) S. Akiyama & H. Ohba and M. sachalinensis (F. Schmidt) S. Akiyama & H. Ohba.

Micranthes fusca occurs in Kyushu, Shikoku, Honshu, Hokkaido and the southern Kuril Islands. It is a perennial with a short rhizome, rounded radical leaves with long petioles, deltoid leaf margins and small actinomorphic dark reddish (sometimes greenish) flowers borne in narrow panicles. Micranthes fusca is divided into two varieties and one form, based on hair types in the inflorescence: var. fusca, with glandular hairs, occurs on Hokkaido and in the northern Tohoku area; var. kikubuki with brown multicellular hairs without clear glands,

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“Japan” by Hamel (1953). For M. japonica, 2n = 28 was reported in an analysis of plants from Shokanbetsu, Hokkaido (Funamoto and Nakamura 1990).

Materials and MethodsWe observed the chromosomes of 22

individuals representing three infraspecific taxa of Micranthes fusca, three individuals of Saxifraga purpurascens Kom. and one individual of M. japonica (Table 1, Fig. 1). The methods for chromosome observation followed Fukuda et al. (2007) with modifications: root tips were incubated in 2mM 8-hydroxyquinoline and stored in 5ºC for 10 h, fixed in Farmer’s fixative at room temperature, then macerated in 1N hydrochloric acid at 60ºC for 10 min., and squashed on the slide after being heated slightly. Karyological terms follow Levan et al. (1964), where type m has arm ratio (length of long arm/ length of short arm): 1.0–1.7, type sm: 1.7–3.0, and type st: 3.0–7.0.

Results and DiscussionThe chromosome number for Micranthes

fusca was 2n = 30 (ca. 30) for three infraspecific taxa, except one individual among the plants of f. kurilensis (Table 1). The chromosomes were 1.6–2.5 µm long, gradually changing its size, and consisted of sm and m pairs. In the karyogram of Fig. 3-a, only two pairs, the 2nd and 3rd, were found to be sm, and all other pairs were m. Satellites were observed on the 2nd pair, as indicated by arrows, though they were not always observed (e.g., arrow in Fig. 2-c). We found satellite-like chromosome in the shorter arm of the 3rd pair, too (Fig. 3-a), but we could not confirm its presence.

A count of 2n = 30 has been reported two times from Hokkaido (Nishikawa 1985) and from Nagano Prefecture, Honshu (Funamoto and Nakamura 1990), and appears to be stable for all the three varieties (Table 1). Plants from Kunashiri and Shikotan of the Kuril Islands also had the same number. Report of 2n = 26, 26–28

occurs in Kyushu, Shikoku and Honshu, except in the Tohoku region; f. kurilensis, without hairs, is distributed from the eastern end of Hokkaido (e.g., Shiretoko Peninsula) to the southern Kuril Islands (Ohwi 1932, 1953, 1965).

Saxifraga purpurascens Kom., occurring in Kamchatka and in the northern Kuril Islands, is considered to be closely related to Micranthes (= Saxifraga) fusca (Zhmylev 1996). It has red petals and small leaves with deltoid margins, and grows on the volcanic slopes. The plant is sometimes treated as a synonym of Micranthes fusca (Hultén 1928, Hara 1939) and not always recognized as an independent species. Although the plant should be recombined with the genus Micranthes in the future, we use the name “Saxifraga purpurascens Kom.” which has been used until now.

Micranthes japonica, with white petals and ovate to cordate radical leaves with irregularly acutely toothed margins, is endemic to Hokkaido, Honshu and Shikoku, Japan (Ohwi 1953).

Cytology of Saxifragaceae and Micranthes has been reported by many authors (e.g., Hamel 1953, Zhukova and Petrovskiy 1987, Wakabayashi and Ohba 1988). The chromosome numbers of Saxifragaceae range from 2n = 10 to 2n = 198 and ca. 220 (Webb and Gornall 1989). Basic numbers has been explained variously, as 10, 11, 13 and 14 (Webb and Gornall 1989). Wakabayashi and Ohba (1988) considered the basic number for Himalayan Micranthes to be x = 11. Polyploidy, followed by an aneuploidy decrease sometimes seems to appear within section Micranthes and others, as counts of 2n = 20, 38, 40 (Webb and Gornall 1989).

The chromosome number for Micranthes fusca was reported to be 2n = 30 in Hokkaido by Nishikawa (1985) and in Nagano Prefecture, central Honshu, by Funamoto and Nakamura (1990). The chromosome numbers 2n = 26 and 26–28 were reported from Kunashiri of the Kuril Islands by Rudyka (in Probatova et al. 2007) and n = 28 and 2n = 56 for plants from

April 2014 TheJournal of Japanese Botany Vol. 89 No. 2 113

from Kunashiri (Rudyka in Probatova 2007) may be erroneous.

We found 2n = 45 for one individual among a population along Stolbovskyy Ecological Road on Kunashiri Island. Karyotypic analysis (Fig. 3-b) suggested that the plant may be triploid, with a basic number x = 15. This individual also had chromosome type of sm for 2nd and 3rd triplets, 13 triplets of m, and had satellites on the 2nd triplet (as indicated by arrows in

Fig. 3-b), although one of the satellites was not clearly observed. Although many plants in the population bore floral buds on their rhizomes, there were no floral buds on the triploid individual. Instead, the plant produced long stolons. During the field observation of this locality we noticed that the plants were widespread in humid coniferous forests, even at long distances from streams. It is possible that such habitat may be related to the occurrence of

Table 1. Locality, sample number and chromosome count for the present study

SpeciesJapanese name Locality no. Locality Sample no.

Somatic chromosome

count Voucher

Micranthes fusca var. fuscaエゾクロクモソウ 1 Hokkaido, Mt. Hirayama SF-1 ca. 30 TI

1 Hokkaido, Mt. Hirayama SF-2 30 TI2 Hokkaido, Kushiro, Akan SF-6 30 TI3 Hokkaido, Kushiro, Kamioboro KAMIOBORO-16 30 TI3 Hokkaido, Kushiro, Kamioboro KAMIOBORO-17 30 TI4 Fukushima pref., E of Mt.

AkatsuraFUKU-8 30 TI

4 Fukushima pref., E of Mt. Akatsura

FUKU-9 30 TI

4 Fukushima pref., E of Mt. Akatsura

FUKU-10 ca. 30 TI

var. kikubukiクロクモソウ 5 Fukushima pref., Azuki spa FUKU-7 30 TI

6 Nagano pref., Mt. Odaka Odaka-3 ca. 30 TI6 Nagano pref., Mt. Odaka Odaka-4 ca. 30 TI6 Nagano pref., Mt. Odaka Odaka-5 ca. 30 TI

f. kurilensisチシマクロクモソウ 7 Hokkaido, Shiretoko, Utoro SF-8 ca. 30 TI

7 Hokkaido, Shiretoko, Utoro SF-9 30 TI8 Hokkaido, Shari, Minehama SF-11 30 TI9 Shikotan Island, Mt. Ploskaya Ploskaya-2 30 TI10 Kunashiri Island, Andreevka KN-2012-5 30 TI11 Kunashiri Island, Stolbovskyy KN-2013-2 30 VLA11 Kunashiri Island, Stolbovskyy KN-2013-4 30 VLA11 Kunashiri Island, Stolbovskyy KN-2013-7 45 VLA11 Kunashiri Island, Stolbovskyy KN-2013-9 30 VLA11 Kunashiri Island, Stolbovskyy KN-2013-11 30 VLA

Saxifraga purpurascens 12 Kamchatka, Mt. Avacha SP-2011-10 24 TI12 Kamchatka, Mt. Avacha SP-2013-2 24 TI*12 Kamchatka, Mt. Avacha SP-2013-6 24 TI*

M. japonica フキユキノシタ 1 Hokkaido, Mt. Hirayama SJ-3 28 TI

* Under procedure.

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triploid plants, because occurrence of M. fusca is usually restricted to the riversides. However, Webb and Gornall (1989) described that saxifrages (= Saxifraga in broad sense, including Micranthes) differ from many other genera in that there is no marked correlation between polyploidy and vegetative reproduction, and careful study will be needed further.

Hamel (1953) reported 2n = 56 for Saxifraga fusca (= M. fusca) growing in the alpine garden in the Göteborg (Gothenburg) Botanical Garden. The origin of the plant was indicated only as “Japan.” Since Hamel precisely observed the size of the chromosomes and arm proportions in gametophytic and somatic cells, his counts cannot be immediately discounted. It would be informative to examine a voucher specimen, but no vouchers were cited in his paper.

The chromosome number of 2n = 24 for

Fig. 1. Sampling localities of Micranthes fusca and its related species. Locality numbers correspond to those in Table 1.

Saxifraga purpurascens Kom. from Kamchatka is the first report for this species. Although we could not get a clear karyogram for this species, we noticed that some of the chromosomes seem to be st-type, having quite off-centered centromeric position (black triangles in Fig. 2-g). Although S. purpurascens is sometimes treated as a synonym of Micranthes fusca (Hultén 1928, Hara 1939), they differ in floral morphology (Fukuda et al. in prep.) and in habitat (Barkalov 2009). The differences in chromosome numbers and karyotypes also suggest that they are distinct.

Chromosome number for Micranthes japonica, 2n = 28, agrees with the previous report (Funamoto and Nakamura 1990).

We would like to acknowledge Dr. D. E. Boufford, Harvard University Herbaria, for his

45°N

40°N

145°E140°E135°E

1 2 3

45

6

7

8

9

10

11

45°N

40°N

145°E140°E135°E

1 2 3

45

6

7

8

9

10

11

50°N

160°E155°E

12

Hokkaido

Honshu

Kamchatka

f. kurilensis

Micranthes fuscavar. fuscavar. kikubukivar. kurilensis

Saxifraga purpurascensMicranthes japonica

50°N

160°E155°E

12

Hokkaido

Honshu

Kamchatka

f. kurilensis

April 2014 TheJournal of Japanese Botany Vol. 89 No. 2 115

Fig. 2. Somatic metaphase chromosomes of Micranthes fusca, Saxifraga purpurascens and M. japonica. a. Micranthes fusca from Mt. Hirayama, Hokkaido (SF-2; 2n = 30). b. M. fusca from Akan, Hokkaido (SF-6; 2n = 30). c. M. fusca from Azuki spa, Fukushima pref. (FUKU-7; 2n = 30). Arrow indicates satellite chromosome. d. M. fusca from Utoro, Shiretoko, Hokkaido (SF-9; 2n = 30). e. M. fusca from Stolbchatyy, Kunashiri Island (KN-2013-2; 2n = 30). f. M. fusca from Stolbchatyy, Kunashiri Island (KN-2013-7; 2n = 45). g. Saxifraga purpurascens from Mt. Avacha, Kamchatka (SP-2013-6; 2n = 24). Black triangles indicate chromosomes with off-centered centromeric position. h. M. japonica from Mt. Hirayama (SJ-3; 2n = 28).

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critical reading and checking English grammar of our manuscript. This study is partly supported by Grants-in-Aid Nos. 21405009 (to H. T.) and B-25292085 (to Dr. Koichi Kaji, Tokyo University of Agriculture and Technology) from the Japan Society for the Promotion of Science.

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des Saxifragacées. Rev. Cyt. Biol. Veg. 14: 113–314.Hara H. 1939. Saxifragaceae. In: Nakai T. and Honda M.

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Fig. 3. Karyograms of Micranthes fusca. a. 2n = 30 (KN-2013-9). b. 2n = 45 (KN-2013-7). Bar = 5 µm.

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Elucidating deep-level phylogenetic relationships in Saxifragaceae using sequences for six chloroplastic and nuclear DNA regions. Ann. Missouri Bot. Gard. 88: 669–693.

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福田知子 a，A. Loguntsevb, I. Bobrovb, M. Antipinb, A. Taranc，高橋英樹 d，池田　博 e：エゾクロクモソウ（ユキノシタ科）と近縁種の染色体数　エゾクロクモソウとその近縁種の染色体数を調べた．エゾクロクモソウ 2変種１品種のうち，2変種（エゾクロクモソウ、クロクモソウ）の染色体数は 2n = 30（または ca. 30）であった．1品種（チシマクロクモソウ）の多くの個体は 2n = 30 であったが，国後島の集団から3倍体（2n = 45）と見られる個体が見つかった．カムチャツカ～千島列島北部に分布し，エゾクロクモソウのシノニムとされることがある Saxifraga purpurascens Kom. の染色体は 2n = 24（初報告）であり，エゾクロ

クモソウとは異なる種であることが示唆された．フキユキノシタの染色体は 2n = 28で，以前の報告が支持された．

（a国立科学博物館植物研究部，b国後島 Kurilsky州立自然保護区，

cロシア・サハリン植物園，d北海道大学総合博物館，

e東京大学総合研究博物館）