_??_1993 The Japan Mendel Society C ytologia 58: 453-461, 1993

Chromosome Studies of Japanese Agrimonia ()

Yoshikane Iwatsubo, Misako Mishima and Noohiro Naruhashi

Department of Biology , Faculty of Science, Toyama University, Gofuku, Toyama 930, Japan

Accepted September 10. 1993

Agrimonia Linn., in the subfamily of the Rosaceae , consists of ca. 15 species which occur in the temperate zone of both the Northern Hemisphere and South America (Airy

Shaw 1973). In Japan, three species and one natural hybrid of Agrimonia: A . coreana, A. nipponica, A. pilosa var. japonica, and A . •~nippono-pilosa, are known (Murata and Umemoto 1983), all of which are in the series Pilosa (Skalicky 1971). Chromosome numbers have been

reported as follows: 2n=28 for A. coreana and A . nipponica, and 2n=56 for A. pilosa var. japonica; whereas that for A. •~nippono-pilosa is not known (Hara and Kurosawa 1968). In addition to further investigation of chromosome numbers for as many localities of

Japanese Agrimonia as possible, this study intends to clarify the phylogenic relationship among

the Japanese Agrimonia by means of karyotype analyses and meiotic chromosome behaviour.

Materials and methods

Chromosome numbers were examined in 455 of Agrimonia, representing three

species and one natural hybrid, collected from 374 localities in Honshu, Shikoku, and Kyushu

in Japan (Table 1). Plants from all localities were grown in the botanic garden of Toyama

University. Newly formed roots were pretreated in a 0.002M 8-hydroxyquinoline solution for

one hour at room temperature and subsequently treated for 15hr at 5•Ž. Fixation was carried out in a fresh mixture of absolute ethanol and glacial acetic acid (3:1) for one hour, and then soaked in 1N HCl at room temperature for a few hours. After being macerated in 1N HCl at

60•Ž for 11.5min, each sample was washed in tap water for more than 3min. Root tips were stained in 1.5% lacto-propionic orcein, and the usual squashing method was employed for the studies of chromosomes. Cells having well spread metaphase chromosomes in several plants from the four taxa were photographed, drawn with a camera lucida, and the chromosome length measured using an Olympus OSM micrometer. The collection localities of the plants whose karyotypes were examined are:

A. coreana Nakai: Higashiyama, Shiojiri-shi, Nagano Prefecture.

A. nipponica Koidz.: Ao, Himi-shi, Toyama Prefecture; Yakushoji-ike, Kosugi-machi, Toyama

Prefecture.

A. pilosa Ledeb. var. japonica (Miq.) Nakai: Manno-cho, Kagawa Prefecture; Furusawa,

Toyama-shi, Toyama Prefecture.

A. •~nippono-pilosa Murata: Nodani, Tokuji-cho, Yamaguchi Prefecture. Chromosome form was expressed according to the nomenclature of Levan et al. (1964).

For studies of meiotic chromosomes, young buds were fixed in Newcomer's fluid at 17•Ž for 3hr and macerated with the same procedure as for the root tips. After being stained with

Schiff's reagent, the anthers were stained and squashed in 1.5% lacto-propionic orcein and chromosome pairing in pollen mother cells (PMCs) was examined. Using more than 2000 grains, pollen fertility was estimated on the basis of the grain size and stainability in lacto propionic orcein. Voucher specimens were deposited in the herbarium of Toyama University. 454 Yoshikane Iwatsubo, Misako Mishima and Naohiro Naruhashi Cytologia 58

Results

(1) Agrimonia coreana Nakai Chromosome numbers have been determined for thirteen plants, collected from two localities (Table 1). All the plants had 2n=28 (Fig. 1A), which was in agreement with the previous report (Hara and Kurosawa 1968). Karyotypes for the two plants from Higashiyama in Nagano Prefecture had no marked differences. The chromosomes at somatic metaphase ranged from 1.1ƒÊm-2.4ƒÊm in length and 1.0 to 2.2 in arm ratio (Table 2). The 28 chromosomes were classified into two groups, eight metacentric pairs, and six submetacentric pairs (Figs. 2A, 5A). Two of the metacentric pairs had satellites on the short arms. The karyotype was formulated as 2n=28=12m+4tm+12sm. Chromosome pairing was examined in 81 PMCs. In all cells, there were 14 bivalents (Fig. 3A, Table 6).

(2) Agrimonia nipponica Koidz. One hundred sixty-three plants from 131 localities have been examined (Table 1). All the plants had 2n=28 chromosomes (Fig. 1B), which confirmed the previous report (Hara and Kurosawa 1968). Karyotypes of two plants from different localities in Toyama Prefecture were nearly the same. The chromosomes at somatic metaphase ranged in length from 1.1ƒÊm-2.5ƒÊm and in arm ratio from 1.0 to 3.2 (Table 3). The somatic chromosome complement was composed of nine metacentric pairs, four submetacentric pairs, and one subtelocentric pair. In one pair of metacentric, and one pair of submetacentric chromosomes, there was a satellite on the short arm (Figs. 2B, 5B). The karyotype was formulated as 2n=28=16m+2tm+6sm+

2tsm+2st. Chromosome pairing was examined at metaphase I in 173 PMCs. All of the cells had 14 bivalents (Fig. 3B, Table 6).

(3) Ledeb. var. japonica (Miq.) Nakai Two hundred seventy-eight plants from 240 localities have been studied. All the plants had

Table 1. List of collection localities and examined plants in each Prefecture of four studied taxa in Japanese Agrimonia 1993 Chromosome Studies of Japanese A grimonia 455

Fig. 1. Somatic metaphase chromosomes of four Japanese taxa of Agrimonia . A: A. coreana, 2n=28; B: A. nipponica, 2n=28; C: A. pilosa var. japonica, 2n=56; D: A. •~nippono-pilosa , 2n= 42. Bar represents 8ƒÊm.

Table 2. Measurements of somatic metaphase chromosomes of Agrimonia coreana

t: satellite

2n=56 chromosomes (Fig. 1C), which is consistent with the report of Hara and Kurosawa

(1968). Karyotypes in the plants from Toyama and Kagawa Prefectures showed no marked differencies. Chromosomes at metaphase ranged from 1.2ƒÊm-2.5ƒÊm in length and 1.0 to 2.5 in arm ratio (Table 4). These were classified into two groups: 21 metacentric pairs, and seven submetacentric pairs. One submetacentric pair had a satellite on the short arm (Figs. 2C, 5C).

The karyotype was thus formulated as 2n=56=42m+12sm+2tsm. Chromosome pairing at metaphase I was examined in 40PMCs. All of them showed 28 bivalents (Fig. 3C, Table 6). 456 Yoshikane Iwatsubo, Misako Mishima and Naohiro Naruhashi Cytologia 58

Fig. 2. Karyotypes of four Japanese taxa of Agrimonia. A: A. coreana, 2n=28; B: A. nipponica, 2n=28; C: A. pilosa var. japonica, 2n=56; D: A. •~nippono-pilosa, 2n=42. Arrows indicate satellite chromosomes. Arrow heads indicate subtelocentric chromosomes. Bar represents 4ƒÊm.

Table 3. Measurements of somatic metaphase chromosomes of Agrimonia nipponica

t: satellite

(4) Agrimonia•~nippono-pilosa Murata One had 2n=42, which is intermediate between A. nipponica and A. pilosa var.

japonica (Fig. 1D). This is the first report of the chromosome number for this taxon. The chromosomes at somatic metaphase ranged from 1.1ƒÊm-2.8ƒÊm in length and 1.0 to 3.2 in arm

ratio (Table 5). There were 30 metacentric chromosomes, 11 submetacentric chromosomes, 1993 Chromosome Studies of Japanese Agrimonia 457

Fig. 3. Meiotic chromosomes at metaphase I in PMCs of three species of Japanese Agrimonia. A: A. coreana, n=14; B: A. nipponica, n=14; C: A. pilosa var. japonica, n=28. Bar represents

10ƒÊ m.

Table 4. Measurements of somatic metaphase chromosomes of Agrimonia pilosa var. japonica

t: satellite and one subtelocentric chromosome (Figs. 2D, 5D). The three chromosomes in the somatic complement, one metacentric and two submetacentric ones, had satellites on the short arms. The karyotype was thus formulated as 2n=42=29m+1tm+9sm+2tsm+1st. Chromosome pairing at metaphase I examined in 96 PMCs, showed 11 to 24 univalents, 9 to 14 bivalents, and 0 to 3 trivalents (Fig. 4, Table 6). The most frequent kind of chromosome pairing was 14II+ 458 Yoshikane Iwatsubo, Misako Mishima and Naohiro Naruhashi Cytologia 58

Table 5. Measurements of somatic metaphase chromosomes of Agrimonia•~nippono-pilosa

t: satellite

Fig. 4. Meiotic chromosomes at metaphase I in PMCs of A . •~nippono pilosa. A: 14II+14I. B: 1III+13II+13I. C: 13II+16I. Arrow indicates trivalent chromosome . Bar represents 10ƒÊm.

14I (45.8%), followed by 1III+13II+13I (11 .5%), 13II+16I (11.5%), etc. The mean chromosome pairing per cell was 0.41III+12 .93II+14.92I. Pollen fertility of this plant was 1.3%, and the plant seldom set seed. By contrast, several plants from each of the three Japanese Agrimonia species showed more than the 98% pollen fertility. 1993 Chromosome Studies of Japanese Agrimonia 459

Fig. 5. Idiograms of four Japanese taxa of Agrimonia. A: Half set of A. coreana (2n=28). B: Half set of A. nipponica (2n=28). C: Half set of A. pilosa var, japonica (2n=56). D: Full set of A. •~nippono-pilosa (2n=42).

Table 6. Chromosome pairing at first metaphase in PMCs of Agrimonia coreana, A. nipponica, A. pilosa var. japonica, and A. •~nippono-pilosa 460 Yoshikane Iwatsubo, Misako Mishima and Naohiro Naruhashi Cytologia 58

Discussion

Chromosome counts of the Japanese Agrimonia were: n=14, 2n=28 for A. coreana and A. nipponica; n=28, 2n=56 for A. pilosa var. japonica; which agrees with the earlier report (Hara and Kurosawa 1968), and 2n=42 for A. •~nippono-pilosa. The 2n=42 count for A. •~ nippono-pilosa is first reported herein. Since the basic chromosome number for Agrimonia has been known to be x=7 (Darlington and Wylie 1955), these counts are interpreted as tetraploid, hexaploid and octoploid levels, respectively. Although A. pilosa var. japonica has twice the chromosome number as A. coreana or A. nipponica, its karyotype indicates that this taxon has not been produced by a simple auto polyploidization of A. coreana or A. nipponica. As shown in Table 4, A. pilosa var. japonica has seven submetacentric pairs. Because three of these pairs (nos. 31-36) are similar in length and four of them vary in length, this species is presumed to be allopolyploid.

Agrimonia•~nippono-pilosa has been considered to be a hybrid between A. nipponica and A. pilosa var. japonica on the basis of the distribution patterns of calcium oxalate crystals in the (Murata and Umemoto 1983). In accordance with the taxonomic interpretation, this plant has an intermediate chromosome number (2n=42) between that of A. nipponica (2n= 28) and that of A. pilosa var. japonica (2n=56). The karyotype formula of this plant was 2n=

42=29m+1tm+9sm+2tsm+1st, which corresponds with half a set of A. nipponica (8m+ 1t m+3sm+1tsm+1st) and half of A. pilosa var. japonica (21m+6sm+1tsm). This taxon is, therefore, thought to be an F1 between A. nipponica and A. pilosa var. japonica. The most

frequent kind of chromosome pairing in PMCs of this hybrid, 14II+14I, provides indirect evidence that A. pilosa var. japonica is an allopolyploid, one of whose putative parents is A. nipponica.

Summary

Four taxa of Japanese Agrimonia were studied cytologically. Chromosome numbers were: n=14, 2n=28 for A. coreana and A. nipponica; n=28, 2n=56 for A. pilosa var. japonica; and

2n=42 for A. •~nippono-pilosa. The karyotypes of the four taxa were gradual and symmetric, and are expressed as follows: A. coreana: 2n=28=12m+4tm+12sm; A. nipponica: 2n=28=

16m+2tm+6sm+2tsm+2st; A. pilosa var.japonica: 2n=56=42m+12sm+2tsm; A. •~nippono pilosa: 2n=42=29m+1tm+9sm+2tsm+1st. The karyotype formula of A. •~nippono-pilosa corresponded with half a set of A. nipponica (8m+1tm+3sm+1tsm+1st) and half of A. pilosa var. japonica (21m+6sm+1tsm). Thus, this plant is thought to be F1 hybrid between A. nipponica and A. pilosa var. japonica. In the PMCs of this proposed hybrid, the most frequent kind of chromosome pairing was 14II+14I, which suggests that A. pilosa var. japonica is an allopolyploid which includes the same genome as that of A. nipponica.

Acknowledgement

The authors are grateful to Mr. H. Masaki for supplying materials from Yamaguchi Prefecture, to Mr. O. Kume for supplying materials from Kagawa Prefecture, and to Dr. Maxine Thompson (National Clonal Germplasm Repository, USDA, USA) for her critical reading of this manuscript.

References

Airy Shaw, H. K. 1973. J. C. Willis's Dictionary of the Flowering Plants and Ferns. 8th ed. p. 84. Cambridge 1993 Chromosome Studies of Japanese Agrimonia 461

University Press, Cambridge. Darlington, C. D. and Wylie, A. P. 1955. Chromosome Atlas of Flowering Plants. p. 141. George Allen and Unwin, London. Hara, H. and Kurosawa, S. 1968. Cytotaxonomical notes on some Asiatic species of Agrimonia. J. Jap. Bot. 43: 392 -400. Levan, A., Fredga, K. and Sandberg, A. A. 1964. Nomenclature for centromeric position on chromosomes. Hereditas 52: 201-220. Murata, G. and Umemoto, K. 1983. Critical notes on the Asiatic Agrimonia with special reference to the distribution patterns of Calcium oxalate crystals. Acta Phytotax. Geobot. 34: 166-177 (in Japanese). Skalicky, V. 1972. Diagnoses, conspectus et claves analyticae taxorum infragenericorum novorum generis Agrimonia L. adhuc rite non publicatorum. Novit. Bot. Univ. Carolinae Pragensis 1971: 13-22.