Cytologia 40: 693-711, 1975
Somatic Chromosome Number and Chromosomal Morphology of Anemone Hepatica L. var. japonica (Nakai) Ohwi
Yutaka Suda
Department of Biology, Iwate University, Morioka , Japan
Received April 30, 1974
Introduction The Anemone Hepatica group of Japan has been revised three times since 1937 (Nakai 1937, Hiroe 1957, Hara 1958). The common attitude among the revisers is to regard the Japanese plants as varieties of the European species. Nakai treated Japanese plants as varieties of the European Hepatica nobilis Schreber (=Anemone Hepatica L.) and divided them into two varieties by the shape of their leaf-lobes, i.e., var. nipponica Nakai having roundish or obtuse leaf-lobes, and var. japonica Nakai having acute leaf-lobes. Hiroe (1957) found a polyploid series among Japanese plants and applied var. japonica (Nakai) Ohwi, var. nipponica (Nakai) Hiroe and var. pubescens Hiroe to diploid, tetraploid and hexaploid plants with the basic number x=7, respectively. According to Hara and Kurosawa (1958), how ever, the relationship between the cytological finding and taxonomical treatment is not so simple and the materials hitherto used for cytological investigations also need further taxonomic examination. They found that the diploid plants (var. japonica (Nakai) Ohwi) had a wide range of distribution, while the tetraploid plants (var. pubescens Hiroe) were restricted to western Honshu and eastern Shikoku. Then, Hara (1958) concluded that the Japanese plants should be regarded as vari eties of the European Anemone Hepatica L. (=Hepatica nobilis Schreber) and di vided them into two varieties and three forms, i.e., var. japonica (Nakai) Ohwi f. japonica, f. magna Hiroe, f. variegata (Makino) Hara and var. pubescens Hiroe. The first report on the chromosome number of the Anemone Hepatica group in Japan was given by Sugiura (1931) as 2n=16. Since then, several authors (Naka jima 1933, Matsuura and Suto 1935, Kurita 1955, 1956, 1957, Hiroe 1957, Hara and Kurosawa 1958 and Suda 1962) reported the chromosome number of the Japanese plants and established the basic number as x=7 in the group. But con fusion has resulted because these authors applied various scientific names to their materials. Following Hara's revision (1958) of the Anemone Hepatica group in Japan, the chromosome numbers hitherto published are summarized in Table 1. Matsuura and Suto (1935) reported brief descriptions on size and centromeric position of somatic chromosomes and a drawing of mitotic metaphase chromo somes of 'Hepatica triloba var. obtusa' based on the permanent slides prepared by the usual paraffin section method. The other karyotype analyses on the group (Kurita 1955, 1956, 1957, Hiroe 1957, Hara and Kurosawa 1958 and Suda 1962) were made by the slides prepared by the squash method with various pretreatments. Therefore, results published to date are not always identical, though some of them 694 Yutaka Suda Cytologia 40 are quite similar to one another (Suda 1962). Only brief reports on the existence of the supernumerary chromosomes in the group were given by Hara and Kurosawa (1958) and Suda (1962); the detailed study has never been undertaken. Distinct morphological variation of the satellite chromosome pair was recog nized in A. Hepatica var. japonica (Suda 1962) but further analysis within a larger scope has remained untouched. In the present study, I attempted to determine the detailed karytype of Anemone
Table 1. Chromosomenumbers of AnemoneHepatica var. japonica published by previousinvestigators
* The original locations are unknown . ** The Japanese name is indicated in parentheses. *** According to Hara and Kurosawa (1958), the plants growing at Mt. Bukozan, where both of them were collected, belong to var. nipponica. **** Two scientific names were applied to the same material which had been cultivated at Matsuyama. The original location is unknown.
Hepatica group in Japan and to report further on the morphological variation of satellite chromosomes. Furthermore, the mode of occurrence of supernumerary chromosomes in each population and intra and inter-individual numerical variation of them within a single population are also presented.
Materials and methods
Two hundred and thirty-one individuals in total were collected randomly from 1975 Somatic Chromosome Number and Chromosomal Morphology of Anemone 695 seven populations in the Tohoku District, i.e., one population in Akita, Iwate and Miyagi Prefs., respectively, and four populations in the Yamagata Pref.; all were transferred to the greenhouse at Iwate University, Morioka, Japan. The localities and the number of individuals for each population are listed in Table 2. Judging from the gross morphology, the plants from Jionji population in Yama gata Pref. and Joge population in Miyagi Pref. are f. variegata (Makino) Hara, while those from other remaining populations, i.e., Juninotaki Fall, Tobishima Island, Oyama populations in Yamagata Pref., Mt. Rokurosan population in Iwate Pref. and Obata population in Akita Pref. are f. magna Hiroe. In spite of such identifications of materials used in the present study, the distinction between these two forms seems rather obscure. To root tips excised from pot-bound plants, four pretreatments were applied: 1) 2-4hours in 0.01% aqueous solution of colchicine at room temperature, 2) 4
Table 2. List of localities of populations, number of plants studied and voucher specimens of Anemone Hepatica var. japonica
* Abbreviation of each population is indicated in parentheses .
hours in 0.002mol/l aqueous solution of 8-hydroxyquinoline at 19-20•Ž, 3) 3hours in saturated aqueous solution of m-bromonaphthalene at 19-20•Ž and 4) 32 hours
in distilled water at 0•Ž. Among them, two pretreatments, by colchicine and by 8-hydroxyquinoline, were applied to all plants examined, while the others were
applied occasionally. The pretreated root tips were fixed for 30minutes in modified Carnoy's fluid
(4 chloroform: 3 absolute ethyl alcohol: 1 gracial acetic acid) and macerated in I normal hydrochloric acid for 20minutes and stained in acetic-orcein for 24 to 48
hours. Temporary slides were made by the squash method and after quick ob servation, good slides were selected and made permanent by a modified McClin
tock's method (McClintock 1929) for further examination. Voucher specimens, slides and photomicrographs are deposited at the Her
barium, Department of Biology, Iwate University, Morioka, Japan (IUM). 696 Yutaka Suda Cytologia 40
Results
All somatic chromosome numbers of the two hundred and thirty-one indivi duals collected from seven populations in the Tohoku District were determined as diploid (2n=14) with the basic number x=7, except for one triploid (2n=21) from the Joge population in Miyagi Pref. Further study on the triploid plant will be reported elsewhere. Thirty-three cells of different individuals from seven populations were selected
Fig. 1. A photomicrograph (a) and an explanatory diagram (b) showing fourteen somatic chro mosomes of Anemone Hepatica var. japonica f. magna pretreated with 0.01% aqueous solution of colchicine for two hours (JH 61568-17, Juninotaki Fall, Yamagata Pref.).
Fig. 2. Somatic chromosomes of A. Hepatica var. japonica f, magna with colchicine pretreatment (2n=14, JH 61568-17, Juninotaki Fall, Yamagata Pref.) arranged by morphological characters. All pairs are quite close to the mean values of chromosome length and of arm ratio obtained from thirty measurements. 1975 Somatic Chromosome Number and Chromosomal Morphology of Anemone 697
for their well-spread metaphase chromosomes to take photomicrographs. The photomicrographs were enlarged at the magnification of 3000•~ and the chromo somes belonging to the longest pair (1st pair) and the shortest satellite chromosome pair (7th pair) in the complement for each photomicrograph were measured for comparison. As the contracting effect of chromosomes by pretreatment is not always constant among the cells even from a single root tip, fifteen of them, among which all the chromosomes belonging to the longest or the shortest salellite chromo some pairs indicate almost similar size in each pair, were chosen for karyotype analysis. Then, using these fifteen photomicrographs, a composite haploid idio
Fig. 3. Composite, haploid idiogram of A. Hepatica var. japonica based on measurements of fifteen diploid cells.
Table 3. Chromosome lengths for composite idiogram of Anemone Hepatica var. japonica (15 cells, 30 measurements indicated)
r=1/s, i=100s/c.
gram was constructed (Figs. 1, 2, 3) with a summary of the arm lengths, the arm ratios and the centromeric indices presented in Table 3. Each arm and total lengths are mean values obtained from thirty measurements. The first and the second pairs are equal in size (11.34ƒÊ), but are easily distin
guished by the distinct difference in arm ratio (1st pair; 1.00, 2nd pair; 1.43). The third and the fourth pairs are also distinguishable with each other in spite of their
equal length (10.00ƒÊ) because their arm ratios are different (3rd pair; 1.00, 4th
pair; 1.14). The fifth pair is a little smaller than the fourth pair in length (9.00ƒÊ), 698 Yutaka Suda Cytologia40
but these two pairs are quite similar and hard to distinguish because their arm ratios are very close to each other (4th pair; 1.14, 5th pair; 1.25). As the sixth pair is smaller than the fifth pair and has apparently a different arm ratio (1.50), it is not
Fig. 4. The variation types recognized in the seventh satellite chromosome pair of A. Hepatica var. japonica. For details see text . 1975 Somatic Chromosome Number and Chromosomal Morphology of Anemone 699 difficult to separate the sixth pair from all others. The shortest seventh pair is also easily distinguished from the other six pairs because of clear chromosomal characters such as a high value of the arm ratio (8.96) and a satellite on the short arm. According to Levan, Fredga and Sandberg's definition of chromosome types by centromeric position (1964), the chromosome complement of Anemone Hepatica var. japonica consists of two pairs of M chromosomes (1st and 3rd pairs) having the centromere at the middle of the chromosome, four pairs of m chromosomes (2nd, 4th, 5th and 6th pairs) having the centromere in the median region, and one pair of t chromosomes (7th pair) having the centromere in the terminal region. No karyo typic difference has been recognized between the two forms, f. magna and f. variegata. On the morphology of the seventh satellite chromosome pair, ten types of variation were recognized by comparing the size of satellite with that of the short arm and of the supernumerary chromosome (Fig. 4). A brief description of the ten types is given below.
type 1: Both satellites are equal in size and are as large as a short arm
(Figs. 4, 5, 6). type 2: Both satellites are equal in size and are larger than a short arm, but smaller than a supernumerary chromosome (Fig. 4). type 3: Both satellites are equal in size and are almost as large as a super numerary chromosome (Figs. 4, 7, 8).
type 1-2: Intermediate between type 1 and type 2. One satellite is as large as a short arm, while the other is larger than a short arm but is
smaller than a supernumerary chromosome (Figs. 4, 9, 10). type 2-3: Intermediate between type 2 and type 3. One satellite is larger than a short arm but smaller than a supernumerary chromosome,
while the other is almost as large as a supernumerary chromosome
(Figs. 4, 11). type 1-3: Intermediate between type 3 and type 1. One satellite is as large as a short arm, while the other is almost as large as a supernumerary chromosome (Figs. 4, 12, 13, 14).
type 1•L: Satellites are different in size from each other. One is as large as a short arm, while the other is smaller, more or less, than a short
arm (Fig. 4). type 1-0: Only one satellite with similar size to short arm exists in the pair ( Figs. 4, 15).
type 2-0: Only one satellite, which is larger than a short arm but is smaller than supernumerary chromosome, exists in the pair (Fig. 4).
type 3-0: Only one satellite, which is as large as a supernumerary chromo some, exists in the pair. This type has never been observed in the
present study (Fig. 4). In addition, other types that cannot be categorized into above ten types are rarely recognized (Fig. 16, Table 4). Based on these ten types of morphological variation for the seventh satellite chromosome pair, the constitution of the seven populations were analyzed. The 700 Yutaka Suda Cytologia 40
Figs. 5-9 and 10-15. Photomicrographs (a) and explanatory diagrams (b) of somatic chromosomes of A. Hepatica var. japonica f. magna (7 and 8, JH 61568-29, type 3; 9, TS 7668-3 , type 1-2; 10 , TS 7668-15, type 1-2; 11, OT 61468-3, type 2-3; 12, OT 61468-10, type 1-3; 13 and 14, TS 7668-22, type 1-3; 15, OS 61670-15, type 1-0) and of A. Hepatica var. japonica f. variegata (5, 1975 Somatic Chromosome Number and Chromosomal Morphology of Anemone 701
JS 62571-6, type 1; 6, JM 52768-15, type 1) with various pretreatments. 5, 7, 9, 10, 11, 12 and 15, colchicine pretreatment; 6, 8-hydroxyquinoline pretreatment; 8, m-bromonaphthalene pretreatment; 13 and 14, low-temperature pretreatment. 702 Yutaka Suda Cytologia 40
Figs. 10-15. Explanation: see in Figs. 5-9. 1975 Somatic Chromosome Number and Chromosomal Morphology of Anemone 703 704 Yutaka Suda Cytologia 40
Fig. 16. Other types of the seventh satellite chromosome pair which cannot be categorized into the ten variation types in Fig. 4. These three were observed rarely in the plants from Mt. Rokuro san, Iwate Pref. and from loge, Miyagi Pref.
Table 4. Frequency of variation type for the 7th satellite chromosome pair in each population
Table 5. Frequency of plants with supernumerary chromosomes in each population
* Percent is indicated in parentheses . 1975 SomaticChromosome Number and ChromosomalMorphology of Anemone 705 results are given in Table 4 and the composite pie-charts are diagrammed in Fig. 17. Plants having supernumerary chromosomes are widely distributing in the Tohoku District (Fig. 18). The present results show that five out of seven popula tions contain plants with supernumerary chromosomes in various degrees (Table 5).
Table 6. Intra-individual numerical variation of supernumerary chromosome in root tip cells in plants from Mt. Rokurosan population, Hanamaki, Iwate Pref.
Intra- and inter-individual variation of number is observed in most of the plants examined (Table 6). Morphologically at least, three types of variation are re 706 Yutaka Suda Cytologia 40
Fig. 17. Pie-charts showing the ratios of plants classified by the variation types of the seventh satellite chromosome pair in each population of A. Hepatica var. japonica from the Tohoku District . 1975 Somatic Chromosome Number and Chromosomal Morph ology of Anemone 707
Fig. 18. Pie-charts showing the ratios of plants with supernumerary chromosomes in each popu lation of A. Hepatica vara japonica from the Tdhoku District. The size of each circle is in proportion 708 Yutaka Suda Cytologia 40 cognized among supernumerary chromosomes. Further examination for super numerary chromosome is now in progress.
Discussion
From the chromosome counts of more than a thousand cells from two hundred and thirty-one plants, it is determined that Anemone Hepatica var. Japonica is diploid with a basic number x=7, confirming the previous counts by several authors (Table 1). Though Sugiura (1931) reported 2n=16 for his material, further as certainment of his count is impossible because he failed to record the original locali ty. Moffett (1932) found in Anemone Hepatica not only diploids with the basic number x=7 but also aneuploids (2n=16) frequently. He explained 2n=16 as the result of somatic doubling of chromosomes. Therefore, it is supposed that there must be aneuploid plants (2n=16) in the Anemone Hepatica group as well. The karyotype presented here is basically similar to that reported by Matsuura and Suto (1935), Hara and Kurosawa (1958) and Suda (1962). The minor differ ence among the previous authors' karyotypic constituents (Matsuura and Suto 1935, Kurita 1955, 1956, 1957, Hara and Kurosawa 1958, Hiroe 1957, Suda 1962) may have arisen chiefly from the various definitions of chromosome types classified by centro meric position by which they based. For instance, Battaglia (1955) limited the concept of median and terminal to mean just these points, indicating arm ratios 1:1 and 0:1, respectively, and by allowing submedian (arm ratio of more than 1:1 but less than 1:3) and subterminal (arm ratio of 1:3 or more) to cover the remaind er of the chromosomes. Levan, Fredga and Sandberg (1964) proposed to use the terms median and terminal in two senses, i.e., as exact points and as regions, which are designated with the capital letters (M and T) and small letters (m and t), respec tively. The four regions, median (m), submedian (sm), subterminal (st) and terminal (t), are to indicate arm ratios (r=1/s) of 1.0-1.7, 1.7-3.0, 3.0-7.0 and 7.0-00, respec tively. Hence, in discussing the difference among the constituents of karyotypes by previous authors, the mere comparison of karyotypic formulae will give mis leading conclusion unless the arm ratio of each chromosome is carefully compared. If Battaglia's definition for representing chromosome types by centromeric position is adopted here, the chromosome complement is changed to show two pairs of chromosomes with a median centromere (1st, 3rd), four pairs of chromosomes with a submedian centromere (2nd, 4th, 5th, 6th) and one pair of chromosomes with a subterminal centromere (7th) in the present material. Based on the thirty measurements of each chromosome, the idiograms re ported previously (Suda 1962) were corrected slightly and the revised haploid idio gram is given in the present paper (Fig. 3). Comparing this idiogram with those of Hepatica nobilis (=Anemone Hepatica), H. americana and H. acutiloba reported by Heimburger (1959), it is demonstrated that the European and the North American species, which are considered taxonomically as very close allies of Anemone Hepatica var. japonica, have almost similar idiograms to that of the Japanese taxon, suggesting a close relation among these four taxa cytologically. Concerning the existence of satellite chromosomes in Japanese taxa , Matsuura 1975 Somatic Chromosome Number and Chromosomal Morphology of Anemone 709
and Suto (1935) first noted that the last pair (7th pair) was characterized by the
presence of a large satellite on each member. Later, their finding was confirmed
by Kurita (1955, 1956, 1957), Hara and Kurosawa (1958) and Suda (1962) . On the other hand, no description or drawing of the satellite chromosomes was given
by Nakajima (1933), Sugiura (1936) and Hiroe (1957). The reason why these reports were antithetic on such a simple question as the presence or absence of
satellite chromosomes may be explained as follows. Firstly, pretreatment and
preparation of material employed by them were different. For instance, colchicine
pretreatment seems, unlike other pretreatments, to have drastic effects of contrac tion of chromosomes so that a satellite on such extremely condensed chromosomes
can be easily overlooked. Secondly, their contrasting results may originate from the character of the satellite itself. The satellite is, sometimes, extremely small in size and hard to recognize (Fig. 4; type 1•L), while in other cases (Figs. 4, types 1-0, 2-0; 15) only one satellite does exist in the pair. In Moffett's drawing (1932), apparent difference in size between the two satel
lites is recognized, though he did not mention this in his account. In my previous
paper (1962) I indicated the morphological variation of the seventh satellite chromo some pair, but detailed observations were not made. From the present data, this variation, together with a slight revision of my previous report (Suda 1962, Fig. 17), has been summarized as shown in Fig. 4. Among ten variation types, three
homomorphic pairs like types 1, 2 and 3, are considered basic, while the other six heteromorphic pairs like three intermediate types (1-2, 2-3, 1-3) and four other types (1-0, 1•L, 2-0, 3-0) may be derived from the basic ones. The three satellite chromosome pairs, i.e., C, D and E of Fig. 17 of my previous report (Suda 1962)
should be corrected to types 3, 3-0 and 1 or 1', respectively. From the pie-charts that resulted from classifying the members of each popu lation by the morphological variation of the seventh chromosome pair, it is apparent
that the two basic types, such as types 1 and 2, and the intermediate and the deriva tive types from them, i.e., types 1-2, 1•L, 1-0 and 2-0, are dominant over the other
three types 3, 1-3 and 2-3 in all populations examined. Comparing the constitu tion of each population, such populational pairs as those from Tobishima Island and Oyama as well as from Joge and Mt. Rokurosan are likely to have a close
resemblance in their constitution (Fig. 17), though the relation between such cyto logical similarity and the gross morphology among these populations remain un
clear. In addition to the previous brief reports on the presence of supernumerary
chromosomes among plants from Oyama, Yamagata Pref. (prov. Uzen) and Sawada, Niigata Pref. (prov. Sado) (Hara and Kurosawa 1958, Suda 1962), the present data emphasize that the supernumerary chromosomes are not uncommon among the
populations examined so far (Fig. 18, Table 5). At present it is too early to discuss the question of whether or not there is a sort of clinal variation in the frequency of
supernumerary chromosomes among populations of Anemone Hepatica var. japo nica; however, it is appropriate to conclude that different populations of this taxon
have their own characteristic mode of frequency of supernumerary chromosomes. 710 Yutaka Suda Cytologia 40
Summary
1. Based on chromosome counts of more than 1000 cells from 231 plants in the Tohoku District, Anemone Hepatica vara japonica is determined to be diploid
with a basic number x=7. 2. Utilizing the Levan, Fredga and Sandberg (1964) definition of chromo some types, the chromosome complement consists of 2 pairs of M, 4 pairs of m and
1 pair of t chromosomes, among which the t chromosomes have satellites (Figs. 1, 2, 3).
3. No karyotypic difference was found between the two forms, f. magna and f. variegata. 4. 10 morphological variations, i.e., 3 basic homomorphic pairs, 3 inter
mediate and 4 derivative heteromorphic pairs from the basic ones, have been re cognized for the seventh satellite chromosome pair (Fig. 4, Table 4). 5. On the basis of analyzing the morphological variation of the seventh satel
lite chromosome pair among 7 populations, it is apparent that 2 basic types (1, 2) and the intermediate and the derivative types of them (1-2, 1•L, 1-0, 2-0) dominate over the other types (3, 1-3, 2-3) (Fig. 17, Table 4).
6. Supernumerary chromosomes are not uncommon among the populations in the Tohoku District; the frequency of plants with supernumerary chromosomes is sometimes high (Fig. 18, Table 5).
7. Intra and inter-individual numerical variation of supernumerary chromo somes is observed in some populations (Table 6).
Acknowledgements
I wish to express my sincere thanks to Dr. Walter H. Lewis of the Department of Biology, Washington University, St. Louis, Missouri, for critical reading of the manuscript and helpful suggestions in preparation of this paper for publication . I am also deeply indebted to the late Mr. Tokuji Araki, Messrs. Kiyohiro Shoji and Ryuzo Kumikawa for their valuable cooperation in collecting materials.
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