Biosystematic Studies on the Genus Polygonatum (Liliaceae) I
_??_1990 by Cytologia, Tokyo Cytologia 55: 443-466 , 1990
BiosystematicStudies on the Genus Polygonatum (Liliaceae) I. Karyotype analysis of species indigenousto Japan and its adjacent regions
Minoru N. Tamura
Department of Botany, Faculty of Science, Kyoto University, Kyoto 606, Japan
Accepted March 9, 1990
The genus Polygonatum (Liliaceae-Polygonateae or Convallariaceae-Polygonateae) includes ca. 58 species and is widely distributed in the northern hemisphere, from the sub tropical to subarctic zone (Bentham and Hooker 1883, Krause 1930, Hutchinson 1934, Dahl gren et al. 1985). According to Tang (1978), the species referred to three series, i.e., Ser. Bracteata, Ser. Alternifolia and Ser. Alte-lobata, occur in Japan, Taiwan and Cheju Island, Korea. The plants of Ser. Bracteata are distributed in northern and northeastern China, Far Eastern U. S. S. R., Korea and Japan, and include 6 species and 2 varieties, among which 5 species and 2 varieties occur in Japan and Cheju Isl. Among the species belonging to this series, chromosome numbers and karyotypes of P. desoulavyi var. desoulavyi and P. involu cratum from China, U. S. S. R. and the Korean Peninsula have been reported (Sokolovskaya 1966, Lee 1967, Abramova 1971, Kim and Kim 1979, Wang et al. 1987), but no reports have been made for the plants in Japan and Cheju Isl. The remaining species of this series are so far unknown karyologically (cf. Table 1). Ser. Alternifolia, which comprises ca. 35 species, is the largest group of the genus, and is distributed in Europe, Asia and North America, among which 8 species, 6 varieties and I form occur in Japan, Taiwan and Cheju Isl. Detailed karyotype analyses have been made on the North American as well as European taxa belonging to this series by various authors (Eigsti 1942, Suomalainen 1947, Therman 1950, Kawano and Iltis 1963b, Nowakowska and Zeglicka 1972, for others see Table 1). However, only scattered information has been avail able for the plants from northeastern Asia. The Bracteata group and the Alternifolia group have been studied taxonomically by Franchet and Savatier (1878), Komarov (1935), Satake (1942), Abramova (1975) and Tang (1978). However, taxonomic concepts of these two groups by the above authors (1. c.) are different, especially as to the identity of P. inflatum. Abramova (1975) used chromosome num bers and karyotypes for the delimitation of these groups in addition to gross morphology. But, her results on chromosome numbers and karyotypes for the Japanese species are different from those of the other authors (cf. Table 1). The purpose of the present study first, is to report the detailed chromosome morphology of all the species belonging to Ser. Bracteata and Ser. Alternifolia occurring in Japan and its adjacent regions through a critical examination of many individuals from different localities, and second, to revise taxonomically these two series based on the results obtained.
Materials and methods
The sources of all materials examined are given in Table 2. The plants collected from their native habitats were transplanted into clay pots and cultivated in the Department of 444 Minoru N. Tamura Cytologia 55
Table 1. Present and previous cytological studies of the genus Polygonatum
* These numbers are first counted . ** Judging from the description of gross morphological characters , this plant should be referred to P. inflatum. 1990 Biosystematic Studies on Polygonatum (Liliaceae) I. 445
Botany, Kyoto University for more than two months . The root tip preparations for the examination of the chromosomes were made by using a modification of the acetic orcein squash method by Kawano and Iltis (1963a) . Excised root tips were stored in 0.2% aqueous colchicine solution at 17•Ž for 4h , then fixed in Farmer's solution for 1-2 min, and subsequently stained in 1% aceto-orcein for ca . 12h, then preserved
in an aceto-orcein (1%) -IN hydrochloric acid (1:1) mixture for a minimum of 15 min . Each of the root tips was transferred onto a glass slide , treated with a drop of acetic acid (45%) glycerine mixture (9:1) for ca. 10 sec, and then gently heated over a flame and squashed. Voucher specimens are preserved in the Herbarium of Kyoto University (KYO) . The karyotype descriptions were made according to Levan et al . (1964). The "basic karyo type" examined in the present study was expressed in terms of homologous chromosome
pairs, and thus some uncertain or unstable secondary or small constrictions and also B-chro mosomes were excluded from the karyotype formula.
Results
Chromosomes at mitotic metaphase were examined (Fig. 1), and the results on each taxon were as follows.
1. Polygonatum cryptanthum Lev. et Van. -2n=18 (Figs. 2 and 9) This is the first count of chromosome numbers for this taxon. Plants collected from two localities, i. e., Mt. Ariake and Kamisaki Cape, in Tsushima Isis., Nagasaki Pref. all proved
to be 2n=18, that is, diploid. The karyotype was composed of one long pair of chromosomes
(ca. 10.5 um in length), six medium-sized pairs (ca. 8.2ƒÊm-ca. 5.9ƒÊm) and two short pairs (ca. 3.9ƒÊm). One pair of the long chromosomes had median centromeres. One of the six medium-sized pairs was also metacentric, but the remaining five pairs were submetacentric. One of the two short pairs had median centromeres, while the other pair was submetacentric. One medium-sized pair of metacentric chromosomes (the 2nd pair) and the other medium
sized pair with submedian centromeres (the 6th pair) had secondary constrictions at the distal regions of the long arms. The karyotypes from the two localities were identical.
2. Polygonatum falcatum A. Gray var.falcatum-2n=18 (Figs. 2 and 9) Plants collected from ten localities (Table 2) were all diploid with 2n=18 chromosomes, which agreed with previous reports (Table 1), although 2n=20 was also reported for this taxon. The karyotypes of these plants were identical with one another, and basically the same as that of P. cryptanthum described above.
3. Polygonatum falcatum A. Gray var. hyugaense Hiyama-2n=18, 2n=18+4B-8B (Figs. 3 and 9) Plants from two localities, Mts. Iwaudo and Sekigan, Kumamoto Pref., were first ex amined karyologically in the present study. Plants from Mt. Iwaudo proved to be 2n=18, diploid, and those from Mt. Sekigan were also diploid, but with B-chromosomes, 2n=18+ 4B-8B. The number of B-chromosomes varied from 4 to 8 in the same individual. The basic karyotype of this variety was identical with that of var. falcatum.
4. Polygonatum involucratum (Franch. et Savat.) Maxim. -2n=18 (Figs. 3 and 9) Plants from four localities, Irimizu, Fukushima Pref., Daibosatsu Pass and Koarama, Yamanashi Pref. and Koidzumi, Shiga Pref. were karyologically examined. All plants were diploid with 2n=18 chromosomes, which is in agreement with previous reports (Table 1). Table 2. Sources of materials 1990 Biosystematic Studies on Polygonatum (Liliaceae) 1. 447 448 Minoru N. Tamura Cytologia 55
However, Lee (1967) reported two different chromosome numbers, 2n=20 and 22, for this taxon from Korea. The basic karyotype of this species was identical with those of P. crypt anthum and P. falcatum.
Fig. 1. Somatic chromosomes of Polygonatum species. A, P. falcatum var. hyugaense from Kuma moto: Mt. Sekigan. B, P. involucratum from Yamanashi: Koarama. C, P. odoratum var. maxi
mowiczii from Hokkaido: Abashiri. D, P. macranthum from Yamagata. E, P. desoulavyi var. azegamii from Tokyo. F, P. miserum from Nagano: Narakawa. G, P. domonense from Fukushima. Detailed localities shown in Table 2. Bar=5ƒÊm. 1990 Biosystematic Studies on Polygonatum (Liliaceae) I. 449
Fig. 2. Somatic chromosomes of Polygonatum species. 1, P. cryptanthum from Tsushima Isis.:
Mt. Ariake; 2, from Tsushima Isis.: Kamisaki Cape. 3, P. falcatum var. falcatum from Yamanashi; 4, from Mie; 5, from Yamaguchi; 6, from Fukuoka; 7, from Ohita; 8, from Kumamoto; 9, from Nagasaki: Mt. Gongen; 10, from Tsushima Isis.: Mt. Shiradake; 11, from Tsushima Isis.: Kami saki Cape; 12, from Cheju Isl. Detailed localities shown in Table 2. Bar=5ƒÊm. 450 Minoru N. Tamura Cytologia 55
Fig. 3. Somatic chromosomes of Polygonatum species. 13, P falcatum var. hyugaense from Kuma
moto: Mt. Iwaudo; 14, from Kumamoto: Mt. Sekigan; 15, P. involucratum from Fukushima; 16, from Yamanashi: Daibosatsu Pass; 17, from Yamanashi: Koarama; 18, from Shiga. 19, P. tri chosanthum from Nagasaki: Mt. Gongen (A); 20, from Nagasaki: Mt. Gongen (B); 21, cultivated in Tokyo Univ. 22, P. humile from Hokkaido; 23, from Aomori. Detailed localities shown in Table 2. Bar=5ƒÊm. 1990 Biosystematic Studies on Polygonatum (Liliaceae) I. 451
Fig. 4. Somatic chromosomes of Polvgonatum species. 24, P. humile from Niigata; 25, from Shiga. 26, P. lasianthum var. lasianthum f. lasianthum from Yamagata; 27, from Fukushima; 28, from Nagano; 29, from Hiroshima; 30, from Okayama. 31, P. lasianthum var. lasianthum f. arnabile from Saga. 32, P. lasianthum var. coreanum from Tsushima Isls.; 33, from Cheju Isl.: Mt. Hanla (A); 34, from Cheju Isl.: Mt. Hanla (B). Detailed localities shown in Table 2. Bar-5ƒÊm. 452 Minoru N. Tamura Cytologia 55
Fig. 5. Somatic chromosomes of Polygonatum species. 35, P. odoratum var. maximowiczii from Hokkaido: Abashiri; 36, from Hokkaido: Haboro; 37, from Hokkaido: Mt. Kannoniwa; 38, from
Hokkaido: Tomakomai. 39, P. odoratum var. pluriftorum from Fukushima; 40, from Ibaraki; 41, from Sado Isl.; 42, from Yamanashi: Koarama; 43, from Nagano; 44, from Fukui: Yokogaki; 45, from Fukui: Awara; 46, from Oki Isis.; 47, from Okayama. Detailed localities shown in Table 2. Bar=5ƒÊm. 1990 Biosystematic Studies on Polygonatum (Liliaceae) I. 453
Fig. 6. Somatic chromosomes of Polygonatum species. 48, P. odoratum var. pluriflorum from Yamaguchi; 49, from Kumamoto; 50, from Cheju Isl.; 51, from Yamanashi: Daibosatsu Pass. 52,
P. odoratum var. thunbergii from Ishikawa; 53, from Fukui: Namimatsu; 54, from Fukui: Gamo. 55, P. cyrtonema from Taiwan: Mt. Ta-tun (A); 56, from Taiwan: Mt. Ta-tun (B). 57, P. inflation from Hiroshima; 58, from Ohita. Detailed localities shown in Table 2. Bar=5ƒÊm. 454 Minoru N. Tamura Cytologia 55
Fig. 7. Somatic chromosomes of Polygonatum species. 59, P. inflatum from Kumamoto; 60, from
Cheju Isl. 61, P. macranthum from Yamagata; 62, from Toyama; 63, from Shiga; 64, from Fuku oka; 65, from Saga. 66, P. desoulavyi var. azegamii from Tokyo. 67, P. domonense from Yamagata. Detailed localities shown in Table 2. Bar=5ƒÊm. 1990 Biosystematic Studies on Polygonatum (Liliaceae) I . 455
5. Polygonatum trichosanthum Koidz. -2n=18 (Figs. 3 and 9) This is the first report of a chromosome number for this rare endemic taxon in Kyushu . Plants from two native sites on Mt. Gongen , Nomosaki in Nagasaki Pref., Kyushu, and those cultivated in the Koishikawa Botanical Garden of the University of Tokyo were karyo logically examined, and were all diploid with 2n=18 chromosomes . The description of this taxon was based on plants cultivated in the Koishikawa Botanical Garden , but the source of these plants is considered to be from Nomosaki and its vicinity in Kyushu. The basic karyo types of the plants examined in this study were all identical with one another , and also agreed with those of P. cryptanthum and P. falcatum. But, it should be noted that a single plant from Mt. Gongen (A in Table 2) had an asymmetric karyotype, one chromosome of the 2nd pair lacking a secondary constriction.
6. Polygonatum humile Fischer -2n=20 (Figs. 3, 4 and 9)
Plants collected from four different localities, Lake Saroma in Hokkaido ; Shiriyazaki Cape, Aomori Pref., Senami, Niigata Pref. and Mt. Ibuki, Shiga Pref. were examined karyo logically, and all proved to possess 2n=20 chromosomes. The results obtained in the present study coincide well with previous reports (Table 1), except for the count made by Kim and Kim (1979) for Korean plants, in which 2n=22 was reported. The basic karyotype was composed of one long pair of chromosomes (ca. 10.7ƒÊm in length), four medium-sized pairs
(ca. 8.3ƒÊm-ca. 7.5ƒÊm) and five short pairs (ca. 6.0ƒÊm-ca. 3.9ƒÊm). One long pair of chromo somes had median centromeres. One of the four medium-sized pairs had median centromeres , and the remaining three pairs were submetacentric. Two of the five short pairs had median centromeres, but the other two pairs were submetacentric and the remaining one pair was subtelocentric. One medium-sized pair of metacentric chromosomes (the 2nd pair) and one medium-sized pair with submedian centromeres (the 5th pair) had secondary constric tions at the distal regions of the long arms. The basic karyotypes of all the plants were identi cal. But, karyotypes of the plants from Lake Saroma, Senami and Mt. Ibuki were asymmetric, one chromosome of the 2nd pair not possessing a secondary constriction.
7. Polygonatum lasianthum Maxim. var. lasianthum f. lasianthum-2n=20, 2n=30 (Figs. 4 and 9) Plants collected from Kaminagawa, Yamagata Pref., Irimizu, Fukushima Pref., Tatezawa. Nagano Pref. and Taishaku-kyo, Hiroshima Pref. were karyologically examined, and proved to possess 2n=20 chromosomes, whereas those from Sashide-jima, a small island in the Inland Sea (Setonaikai) in Okayama Pref. were triploid with 2n=30 chromosomes. The basic karyo type of this species resembled that of P. humile, but it was slightly different from the latter in the following respects: (1) the 6th pair was shorter and the 8th pair longer than those of P. humile, (2) centromeres of the 6th and the 7th pairs were located more proximally than those of P. humile, and thus the 6th pair of P. lasianthum had median centromeres, although this pair of chromosomes had submedian centromeres in P. humile. The basic karyotypes of the plants from the five different localities were all identical. Judging from the chromosome morphology, the plants from Sashide jima are assumed to be autotriploid.
8. Polygonatum lasianthum Maxim. var. lasianthum f. amabile (Yatabe) Makino-2n=20 (Figs. 4 and 9) A single plant collected from Mt. Seburi, Saga Pref. was examined karyologically, and proved to be diploid with 2n=20 chromosomes. Its basic karyotype was identical with that of f. lasianthum. 456 Minoru N. Tamura Cytologia 55
9. Polygonatum lasianthum Maxim. var. coreanum Nakai-2n=20 (Figs. 4 and 9) Plants from two localities, i.e., Mt. Shiradake, Tsushima Isis., Nagasaki Pref., and Mt. Hanla (A and B from two different sites), Cheju Isl., Korea, were examined karyologically. All plants possessed 2n=20 chromosomes and had identical basic karyotypes. However, those from Mt. Hanla (A and B) had asymmetric karyotypes, one chromosome of the 2nd pair did not have a secondary constriction. The basic karyotype of var. coreanum was iden tical with that of var. lasianthum.
10. Polygonatum odoratum (Mill.) Druce var. maximowiczii (F. Schmidt) Koidz. -2n=20
(Figs. 5 and 9) Polygonatum odoratum, a wide-ranging Eurasian species, is exceedingly polymorphic.
Three different varieties are distributed throughout the Japanese Islands and its vicinity, al though the type variety, var. odoratum, does not occur in this region. In the present study, all three varieties, var. maximowiczii, var. pluriflorum and var. thunbergii were karyologically examined. Plants of var. maximowiczii collected from four different localities in Hokkaido
(Abashiri, Haboro, Mt. Kannoniwa and Tomakomai) were studied. All plants were diploid, 2n=20. The karyotype was composed of one long pair of chromosomes (ca. 8.7ƒÊm in length), five medium-sized pairs (ca. 7.4ƒÊm-ca. 5.9ƒÊm) and four short pairs (ca. 5.5ƒÊm-ca. 3.8ƒÊm). One long pair of chromosomes had median centromeres, and three of the five medium sized pairs were also metacentric, while the remaining two pairs were submetacentric. Three of the four short pairs had median centromeres, and one pair was submetacentric. One pair
of the metacentric short chromosomes (the 8th pair) had secondary constrictions at the cent ral region of the long arms. The basic karyotypes were identical in all plants from the four localities. The plants from Haboro, Mt. Kannoniwa and Tomakomai possessed one medium sized metacentric pair of chromosomes (the 2nd pair) with secondary constrictions at the
proximal region of the long arms. However, in those from Abashiri, such proximal secondary constrictions were lacking. It should be also noted here that in the plant from Haboro, a small constriction was observed in the distal portion of the long arm of one chromosome of the 3rd pair, which was, however, often invisible in the same individual in some preparations.
11. Polygonatum odoratum (Mill.) Druce var. pluriflorum (Miq.) Ohwi -2n=20, 2n=30 (Figs. 5, 6 and 9) Plants collected from 13 localities (see Table 2) were karyologically examined, of which those from 12 localities were diploid with 2n=20 chromosomes, while a single plant from one locality, Daibosatsu Pass, Yamanashi Pref., was turned out to be 2n=30, triploid. With the exception of the triploid, the present results agreed with those of previous studies (Table 1). This triploid chromosome number was detected in this taxon for the first time. The basic karyotypes of all plants were identical with one another and also with that of var. maximo wiczii. But, in the plant from Sakiyama Cape, Oki Isls., Shimane Pref., one chromosome of the 2nd pair had a secondary constriction at the proximal region of the long arm. Karyo types of plants from the remaining 12 localities were all symmetrical. Judging from the chro mosome morphology, the plant from Daibosatsu Pass is considered to be autotriploid.
12. Polygonatum odoratum (Mill.) Druce var. thunbergii (Morr. et Decne.) Hara-2n=20 (Figs. 6 and 9) Plants from three localities, Chirihama, lshikawa Pref. and Namimatsu and Gamo, Fukui Pref. were karyologically investigated, and all possessed 2n=20 chromosomes. This is the first chromosome count for this taxon. The basic karyotypes of the plants from the above three localities were identical with one another, and also agreed with that of var. maximowiczii. 1990 Biosystematic Studies on Polygonatum (Liliaceae) I. 457
However, karyotypes of the plants from Chirihama and Namimatsu were asymmetric, only one chromosome of the 2nd pair possessing a secondary constriction at the proximal region of the long arm.
13. Polygonatum cyrtonema Hua-2n=22
(Figs. 6 and 9) Plants collected from two sites on Mt . Ta-tun, Taiwan were studied, and proved
to be diploid with 2n=22 chromosomes. Previously, two different numbers, 2n=22 and 44, were reported for this taxon (Hsu
1971, Chang and Hsu 1974, Wang et al . 1987). The basic karyotype of this species had no long chromosomes with median centromeres, but was composed of six pairs of medium-sized chromosomes (ca. 9.9ƒÊm
ca. 9.1ƒÊm in length) and five short pairs
(ca. 6.6ƒÊm-ca. 4.6ƒÊm). Three of the six medium-sized chromosome pairs had median
centromeres, and the remaining three pairs were submedian. Three of the five short
pairs were median, and the remaining two pairs were submedian. One pair of medium sized chromosomes with median centromeres
(the 1st pair) had secondary constrictions at the distal region of the long arms, and one pair of median short chromosomes (the 7th pair) had secondary constrictions at the
proximal region of the long arms. The basic karyotypes of the plants from two Fig. 8. Somatic chromosomes of Polygonatum species. 68, P. domonense from Fukushima. 69, localities were identical, although a single P. miserum from Nagano: Mitake; 70, from Naga plant from Mt. Ta-tun (A) had an asym no: Mt. Hakamagoshi; 71, from Nagano: Nara metric karyotype, one chromosome of the kawa. Detailed localities shown in Table 2. Bar 4th pair having a secondary constriction =5ƒÊm. in the proximal portion of the short arm.
14. Polygonatum inflatum Komar. -2n=22 (Figs. 6, 7 and 9) Plants collected from four localities, Taishaku-kyo, Hiroshima Pref., Inoseto, Ohita Pref., Shimizu Pass, Kumamoto Pref. and Mt. Hanla, Cheju Isl., Korea, were examined karyological ly, and proved to possess 2n=22 chromosomes, which agreed with previous reports (Table 1). The basic karyotype, which was different from that of P. cyrtonema, had no long chromosomes with median centromeres, but was composed of five medium-sized pairs of chromosomes
(ca. 9.1ƒÊm-ca. 7.8ƒÊm in length) and six short pairs (ca. 5.6ƒÊm-ca. 4.1ƒÊm). One of the five medium-sized chromosome pairs had median centromeres, another pair was submedian and the remaining three pairs were subterminal. Two of the six short pairs were median, the other two pairs were submedian and the remaining two pairs were subterminal. One medium sized pair with median centromeres (the 1st pair) and one medium-sized pair with submedian 458 Minoru N. Tamura Cytologia 55
Fig. 9. Karyograms showing the somatic karyotypes of Polygonatum species. Each thick ideogram shows a homologous chromosome pair, and each thin ideogram shows a single chromo some. Two thin ideograms standing abreast show the existence of an asymmetric chromosome pair: a homologous chromosome pair in black thin ideogram shape and an asymmetric chromo some pair in shape of a black thin ideogram and a white thin ideogram with black dots. Small black squares show B-chromosomes, although the numbers vary. For details see the text. 1990 Biosystematic Studies on Polygonatum (Liliaceae) I. 459 centromeres (the 4th pair) had secondary constrictions at the distal regions of the long arms. The basic karyotypes from the four localities were identical. But, karyotypes of the plants from Taishaku-kyo and Inoseto were asymmetric, one chromosome of the 1st pair lacking the secondary constriction.
15. Polygonatum macranthum (Maxim.) Koidz. -2n=22, 2n=22+1B, 2n=22+3B (Figs. 7 and 9) Plants collected from Kaminagawa, Yamagata Pref. and Mt. Seburi, Saga Pref. possessed 2n=22 chromosomes, which agreed with previous reports (Table 1), while those from Hanaore Pass, Shiga Pref. proved to possess 2n=22+1B chromosomes, which agreed with Kawakami (1958) and Jinno (1962), and those from Shimosasahara, Toyama Pref. and Mt. Wakasugi, Fukuoka Pref. proved to be 2n=22+3B, a new count. The number of B-chromosomes was constant in the same individual. The basic karyotype of this species resembled that of P. inflatum, but it was slightly different from the latter in the following respects: (1) the 5th and the 9th pairs of chromosomes were slightly shorter and the 6th pair much longer than those of P. inflatum, (2) centromeres of the 7th and the 8th pairs were located more proximally than those of P. inflatum, and thus the 7th pair of P. macranthum turned out to have submedian centromeres and the 8th pair median centromeres, though the 7th and the 8th pairs of P. inflatum had subterminal and submedian centromeres, respectively. The basic karyotypes of the plants from the five localities were identical. But, the plants from Mt. Wakasugi had an asymmetric karyotype, one chromosome of the 1st pair lacking secondary constriction. It should be noted also that in the plant from Shimosasahara, a small constriction was observed in the central portion of the long arm of one chromosome of the 5th pair, which was, however, often invisible in the same individual in some preparations.
16. Polygonatum desoulavyi Komar. var. azegamii Ohwi -2n=18 (Figs. 7 and 9) This is the first report of a chromosome number for this rare taxon. Plants collected from Hinohara, Tokyo, were examined karyologically, and proved to be 2n=18. Its basic karyotype was almost identical with that of P. cryptanthum, but two chromosomes which se emed to form a pair were highly unequal in length, i. e., its karyotype was quite asymmetric.
17. Polygonatum domonense Satake-2n=19 (Figs. 7-9) This is the first report of a chromosome number for this rare taxon. Plants collected from Ohmaki, Yamagata Pref. and Irimizu, Fukushima Pref. were karyologically examined, and proved to possess 2n=19 chromosomes. The basic karyotype resembled that of P. miserum, but it differed from the latter in the following respects: (1) just as was found in P. desoulavyi var. azegamii, two chromosomes which seemed to form a pair were unequal in length, thus conspicuously asymmetric, (2) compared with those of P. miserum, the chromo some marked with in Fig. 9 was relatively shorter, and the chromosome
18. Polygonatum miserum Satake-2n=19 (Figs. 8-9) This is the first report of a chromosome number for this rare taxon. Plants collected from three different localities in Nagano Pref. (Mitake, Mt. Hakamagoshi and Narakawa) were examined karyologically, and proved to be 2n=19. The karyotype was composed of eight 460 Minoru N. Tamura Cytologia 55
pairs of chromosomes and three unpaired chromosomes. One pair was long (ca. 9.5ƒÊm in length), four pairs were medium-sized (ca. 7.5ƒÊm-ca. 6.5ƒÊm) and three pairs were short (ca. 5.0ƒÊm-ca. 3.7ƒÊm). One unpaired chromosome was medium-sized (ca. 6.5ƒÊm) and two unpaired ones were short (ca. 4.6ƒÊm-ca. 3.8ƒÊm). One long pair of chromosomes had median centromeres. One of the four medium-sized pairs was median and the remaining three pairs were submedian. One of the three short pairs was median and the remaining two pairs were submedian. The unpaired medium-sized chromosome was submedian and the unpaired short ones were median and subterminal, respectively. One pair of medium-sized chromo somes with median centromeres and one pair of medium-sized chromosomes with submedian centromeres had secondary constrictions at the distal regions of the long arms. The karyo types from the three localities were identical.
Discussion
The chromosome numbers of the following Polygonatum taxa were reported for the first time in the present study: 2n=18 for P. crvptanthum, P. desoulavyi var. azegamii, P. falcatum var. hyugaense and P. trichosanthum, 2n=18+4B-8B for P. falcatum var. hyugaense, 2n=19 for P. domonense and P. miserum, 2n=20 for P. lasianthum var. lasianthum f. amabile and P. odoratum var. thunbergii, 2n=22+3B for P. macranthum, and 2n=30 for P. lasianthum var. lasianthum f. lasianthum and P. odoratum var. pluriiorum (see Table 1). For other taxa, previous counts as shown in Table 1 were confirmed. As a result, it became clear now that at least three different basic chromosome numbers, i.e., x=9, 10 and 11, are present in plants referred to the genus Polvgonatum that occur in Japan and its adjacent regions. However, several other basic chromosome numbers, such as x=12, 13, 14, 15, 16, 18 and 19 are also known from plants which occur in Europe, U. S. S. R., the Himalayas, N. Indo-China, China and Korea (Berg 1933, Tischler 1935, Suoma lainen 1947, Therman 1953, Kumar 1959, Mehra and Pathania 1960, Arora 1961, Abramova 1965, 1971, Kurosawa 1966, Polatschek 1966, Sokolovskaya 1966, Lee 1967, Lovka et al. 1971, 1972, Mehra and Sachdeva 1971, 1976, Skalinska et al. 1971, Malla et al. 1981, Love and Love 1982, Wang et al. 1987, Yang et al. 1988, Tamura, unpubl.). All the evidences available in dicate quite complicated cytogenetic differentiation among the taxa referred to the genus Polygonatum. Jeffrey (1980) in his taxonomic revision of the genus Polygonatum regarded P. cryptant hum, P. desoulavyi var. azegamii and P. miserum as conspecific with P. involucratum. His species concept, however, sharply contradicts earlier taxonomic treatments and also my own current conclusion. According to the present study, the karyotype of P. cryptanthum is iden tical with that of P. involucratum (Figs. 2 and 3), although these two taxa are considered to be discernible entities. But, the karyotypes of P. desoulavyi var. azegamii and P. miserum were entirely different from each other and from that of P. involucratum (Figs. 3, 7, 8 and 9). The karyotypes of P. desoulavyi var. azegamii, P. domonense and P. miserum are so het erogeneous in composition that they may possibly be structural hybrids or hybrid derivatives between the two taxa, i.e., P. involucratum and P. falcatum var. falcatum, P. involucratum and P. lasianthum var. lasianthum, P. involucratum and P. humile, respectively (Figs. 7-9). The species examined in the present study are classified into five groups based on chro mosome numbers and basic karyotypes, i.e., the Involucratum-group, the Humile-group, the Odoratum-group, the Inflatum-group and the Cyrtonema-group. P. cryptanthum, P. falcatum, P. involucratum and P. trichosanthum possess identical basic karyotypes with x=9 chromosomes (Figs. 2, 3 and 9), and thus are classified in the Involucratum-group, which has 1990 Biosystematic Studies on Polygonatum (Liliaceae) I . 461
the basic karyotype* of x=9 (n)=1M+1M#+4SM+ 1SM#+1m+lsm . P. humile, P. lasianthum and P. odoratum possess x=10 chromosomes . Jinno (1962) pointed out the dis similarity between the karyotypes of P. humile and P. odoratum, while Inoue (1965) demon strated a similarity between the karyotypes of P. humile and P. lasianthum. Their results were confirmed by the present study (Figs. 3, 4, 5, 6 and 9). Accordingly, there is no doubt that P. humile and P. lasianthum belong to the same group , i. e., the Humile-group, which has the basic karyotype* of x=10 (n)=IM+1M#+2SM+1SM#+2m (or 3 m)+2sm (or 1sm)+ 1st. Although it has x=10 as the basic number , P. odoratum obviously belongs to another group, i.e., the Odoratum-group, which has the basic karyotype* of x=10 (n)=1M+3M (or 2M+1M#)+2SM+2 m+lm#+1sm. P. cyrtonema, P. inflatum and P. macranthum were found to possess x=11 chromosomes. The karyotypes of P . inflatum and P. macranthum resemble each other, but are different from that of P. cyrtonema (Figs. 6, 7 and 9). Accord ingly, P. inflatum and P. macranthum belong to the same group, i.e., the Inflatum-group, with x=11 (n)=lM#+1SM#+3ST+2m (or 3m)+2sm+2 st (or 1st) . P. cyrtonema belongs to another group, i.e., the Cyrtonema-group with x-11 (n)=2M+M#+3SM+2m+lm# +2sm. Suomalainen (1947) distinguished two main karyotypes in Sect. Alternifolia (including Ser. Bracteata and Ser. Alternifolia), i.e., the Latifolium-type with two long pairs of SAT chromosomes and the Officinale-type with one long pair and one short pair of SAT-chromo somes. If the grouping of Suomalainen (1947) is applied to the present results, the following eight species, i.e., P. cryptanthum, P. falcatum, P. involucratum, P. trichosanthum, P. humile, P. lasianthum, P. inflatum and P. macranthum belong to the Latifolium-type, and P. odoratum var. maximowiczii and P. cyrtonema to the Officinale-type. However, P. odoratum var. plu riflorum and var. thunbergii belong to neither type. All of the karyological evidence presented, as well as recent observations on micro-morphological features of filaments by SEM (Tamura , unpubl. and MS in preparation), clearly indicate that the subgroupings of the species from Japan, Taiwan and Cheju Isl. based only on karyotypes, and disregarding the basic chromo some numbers (cf. Suomalainen 1947), are insufficient for the delimitation of infrageneric taxa. Franchet and Savatier (1878) distinguished the species with bracts from those lacking bracts, and established a genus Periballanthus for the former. Satake (1942) reduced them to subgeneric rank under the genus Potygonatum, i.e., Subgen. Periballanthus and Subgen. Eupolygonatum. Abramova (1975) monographed the genus Polygonatum, and recognized three sections in the genus, i.e., Sect. Polygonatum, Sect. Verticillata and Sect. Oppositifolia, and furthermore, under Sect. Potygonatum five series were distinguished including Ser. Brac teata with bracts and Ser. Polygonatum lacking them. In addition to gross morphological characters, she also attempted to use chromosome numbers and karyotypes for the delimita tion of infrageneric taxa. In 1975, Abramova reported x=10 for P. falcatum, x=9 for P. humile and x=10 for P. macranthum, respectively, and further classified the karyotype of P. lasianthum in the Odoratum-type. However, the results obtained in the present study were different, i.e., x=9 was found for P. falcatum, x=10 for P. humile, and x=11 for P. mac ranthum, respectively, and the karyotype of P. lasianthum can be classified in another group, i.e., the Humile-group. One of the key characters which Abramova used for delimiting infrageneric taxa was the presence or absence of bracts, just as was done by Satake (1942) (Fig. 10). But, the following two authors (Komarov 1935, Tang 1978) regarded the size or
* On the formulas of basic karyotypes used in the present study , (1) bold capitals, capitals and small let ters indicate long, medium-sized and short chromosomes respectively, (2) m, sm and st indicate median, sub median and subterminal centromeric chromosomes respectively, (3) numerals indicate numbers of pairs of chromosomes, (4)# indicates chromosomes with secondary constrictions, (5) () indicates the other choices. 462 Minoru N. Tamura Cytologia 55 the texture of bracts as a significant character of diagnostic value. Komarov (1935) estab lished Ser. Bracteatae with large bracts and four other Series lacking large bracts. However, several species that occur in Japan, Taiwan and Cheju Isl. which lack large bracts belong to none of Komarov's Series according to his key. Tang (1978) recognized Komarov's Ser. Bracteata with large herbaceous bracts, in addition to seven other Series lacking large her baceous bracts, including Ser. Alternifolia, to which all the species without large herbaceous bracts examined in the present study belong (Fig. 10).
Fig. 10. Diagrams demonstrating the inter-relationships between species based on basic chromo some numbers, basic karyotypes and characters of bracts in Polygonatum.
Accordingly, P. cryptanthum and P. involucratum with large herbaceous bracts were classified in Ser. Bracteata by Abramova (1975) and in Ser. Bracteata by Tang (1978). P. falcatum, P. trichosanthum, P. humile, P. lasianthum, P. odoratum, P. macranthum and P. cyrtonema lacking bracts were classified in Ser. Polygonatum by Abramova (1975) and in Ser. Alternifolia by Tang (1978). P. inflatum with small membranous bracts was classified in Ser. Bracteata by Abramova (1975), but in Ser. Alternifolia by Tang (1978) (Fig. 10). 1990 Biosystematic Studies on Polygonatum (Liliaceae) I. 463
The chromosome number (2n=22) and the karyotype of P . inflatum resemble those of P. macranthum, but are different from those of P . cryptanthum and P. involucratum. Accordingly, the results obtained in the present study contradict Abramova's treatment (1975) in which P. inflatum was separated from the ebractiate species and grouped in the species with large herbaceous bracts (Fig. 10). According to the concepts of Krause (1930) and Hutchinson (1934) , the tribe Polygonateae consists of nine genera, i. e., Polygonatum , Disporopsis, Maianthemum, Smilacina**, Oli gobotrya***, Drymophylla, Streptopus, Disporum and Clintonia, although Dahlgren et al. (1985) have recently proposed entirely different systems, only three genera, Polygonatum, Maianthemum and Smilacina, referring to Convallariaceae-Polygonateae , and all other genera referring to different families, e. g., Uvulariaceae-Uvularieae (cf. Bentham and Hooker 1883). The karyotypes of Smilacina and Maianthemum were previously examined by various authors , i. e., Mehra and Pathania (1960) for S. purpurea, Kawano and Iltis (1963a, 1966) for S. race mosa, S. stellata, S. trifolia and S. paniculata, Hara and Kurosawa (1963) for S. japonica and S. yezoensis, Kawano (1965b) for S. scilloidea, Kurosawa (1966) for Sa fusca and S. purpurea, Chang and Hsu (1974) for S. formosana, Mehra and Sachdeva (1979) for S. oleracea, Kawano (1965a) for M. canadense, Kawano et al. (1967, 1971) for M. bifolium, M. dilatatum and M. canadense. But, the basic karyotypes (x=18) for all taxa belonging to these two genera were similar to one another (cf. Kawano et al., 1967). Utech and Suda (1975) and Utech (1975) reported almost the same karyotypes (x=14) for Clintonia udensis, C. andrewsiana, C. umbellulata, C. borealis (cf. Kawano 1965a) and C. uniflora, although they reported also x=16 chromosomes for one clone of C. borealis. The karyotypes (x=20) of Disporopsis arisanensis and D. fiasco pitta were previously studied by Chang and Hsu (1974) and Kumar and Brandham (1980), respectively, and the karyotypes of these two taxa were almost identical. The karyotypes (x=8) of Streptopus amplexifolius and S. roseus were examined by Bent and Smith (1969), and shown to resemble each other. However, from Disporum, various karyo types with x=7, 8, 9 and 11 chromosomes were reported by Hasegawa (1932), Washiashi (1935), Kayano (1960), Mehra and Pathania (1960), Chao et al. (1963), Kurosawa (1966), Arano and Nakamura (1967), Fujishima and Kurita (1973), Chang and Hsu (1974), Noguchi and Kawano (1974), Utech and Kawano (1974, 1977), Mehra and Sachdeva (1976) and Tamura and Kawano (unpubl.). Chromosome number (x=10) of Drymophylla has so far been re ported only by Jackson (1955), but the karyotype of this taxon is not known yet. For Po lygonatum, as was described in the preceding section of this paper, various chromosome num bers (x=9, 10, 11, 12, 13, 14, 15, 16, 18 and 19) and karyotypes are known to occur, just as in the case of Disporum; especially, Ser. Bracteata and Ser. Alternifolia have proved to pos sess not less than five different basic karyotypes (x=9, 10 and 11). It should be noted here that the basic karyotypes of the genera referred to the Polygo nateae sensu Krause (1930) are remarkably uniform, except for two genera, Polygonatum and Disporum. We do not know precisely yet, however, the origin of such diverse karyological differentiations found in Polygonatum and Disporum, which sharply contrasts with the modes detected in some other members of the tribe. In order to understand better their origin and true affinities, more comprehensive karyological as well as molecular analyses are definitely needed.
Summary
Thirteen species, including six varieties and one form, of the genus Polygonatum of Japan,
** LaFrankie (1986a, b) included Smilacina in a single genus, Maianthemum. *** Hara (1975) regarded recently Oligobotrya to be congeneric with Smilacina. 464 Minoru N. Tamura Cytologia 55
Taiwan, and Cheju Island, Korea, were examined karyologically. Among the species with x=9, P. cryptanthum, P. involucratum and P. trichosanthum possessed 2n=18 chromosomes, while Pmfalcatum was 2n=18 and 2n=18+4B-8B. All four species possessed almost iden tical karyotypes. While, among the species with x=10, P. humile possessed 2n=20 chromo somes. Diploid (2n=20) and triploid (2n=30) plants were found for P. lasianthum and P. odoratum. Those with 2n=30 chromosomes are considered to be autotriploid. Karyo types of P. humile and P. lasianthum resembled each other, but were quite different from that of P. odoratum. Among the species with x=11, P. inflatum and P. cyrtonema possessed 2n=22, and P. macranthum was 2n=22, 2n=22+1B and 2n=22+3B. Karyotypes of P. inflatum and P. macranthum resembled each other, but were quite different from that of P. cyrtonema. Karyotypes of P. desoulavyi var. azegamii with 2n=18, P. domonense with 2n= 19 and P. miserum with 2n=19 were quite asymmetric, thus not forming homologous pairs. The origin of such heterogenous karyotypes is not clear at present. Based on these results, the status of each taxon and previous taxonomic treatments were discussed.
Acknowledgements
I extend my cordial gratitude to Prof. Shoichi Kawano for his guidance throughout the study, and for his invaluable comments on the first draft of the paper. My thanks are due to Dr. Hiroshige Koyama, Mr. Gen Murata and Dr. Junko Noguchi for their comments on this work, and to Prof. William F. Grant for his critical reading of the manuscript. Thanks are also due to all the members of the Plant Taxonomy section for their ardent encouragement and discussions at various stages of the study.
References
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