C2001 The Japan Mendel Society Cytologia 66: 105-111, 2001

The Origin and Evolution in Trillium 1. The Origin of the Himalayan Trillium govanianum

Ichiro Fukuda

The Asian Ecology-Evolution Institute, Tokyo 192-0913, Japan

Accepted January 18, 2001

Summary By means of morphological and chromosomal analyses it is considered that the Hi- malayan species Trillium govanianum is an intergeneric hybrid between Trillium and Daiswa. Trilli- um govanianum is an allotetraploid (4x=20) that most likely has originated from the 10 GG genome of a plant in the genus Trillium and the 10 DD genome of a plant from the genus Daiswa. Factors concerning the emergence of the polyploid species have been discussed from an evolutionary view- point and on a consideration of the paleoclimatics in the Himalayan mountain region. Key words Himalayan Trillium, Intergeneric hybrid, Polyploid speciation, Pleistocene survival.

Trillium govanianum Wallich ex Royle (Trilliaceae) is distributed scantily in Kashmir, Nepal,

Sikkim, Darjeeling and Bhutan throughout the Himalayan mountain range. The plants grow at an

elevation of 2800-3960 m under Abies spedtabilris, Tsuga dumosa and Rhododendron forests at longitude 73-92•KE, latitude 27-30•KN. This habitat distribution records the growing region for the

most western edge and the highest elevation for species in the genus Trillium. The chromosome number of T govanianum, 2n =4x=20, has been clearly documented by Haga and Watanabe

(1966), Kurosawa (1971), Chatterjee (1973), Mehra and Sachdeva (1976, 1977) and Kurmar and Subramaniam (1989). The purpose of this article is to analyze in greater detail the chromosome banding pattern of T govanianum and determine if the original diploid chromosomes may be re-

solved by the cold-induced banding method that was adopted for the Japanese and American

species (Darlington and La Cour 1940, Haga and Kurabayashi 1953, Fukuda 1984). Moreover, to aid biosystematically in ascertaining the original parental species of T govanianum, comparative

morphological analyses were carried out with related species comparing floral organs and leaf mor-

phology. Finally, evolutionary and background data will be discussed on the role of polyploid speci- ation in Trillium in Asia.

Materials and methods

Materials of T govanianum were collected at Gosainkund in Nepal (for the cytological investi-

gation) and Chiley-La, Bhutan (for morphological data). Materials for the related species, Daiswa

polyphylla, were collected at Phakding in Nepal. Materials for the related species Trillium tschonoskii were collected at Yahiko, Niigata in Japan. Chromosome data for D. polyphylla and T

tschonoskii were obtained from Fukuda and Peng (1999), Haga and Kurabayashi (1953) and Fukuda and Kan (1966).

Chromosome investigations on T govanianum, D. polyphylla and T tschonoskii were carried out on root-tip cells after cold treatment (0•Ž) for 96 h. The root-tips were fixed in La Cour 2BE for

15 min, hydrolyzed in 1N HCl at 60•Ž for 15 min and stained in Feulgen solution for 60 min. Squash preparations were made in 45% acetic acid. The heterochromatin-banding patterns were de-

termined by using camera lucida drawings from the chromosome preparations. Darlington and La

e-mail: [email protected] 106 Ichiro Fukuda Cytologia 66

Cour (1938) who first discovered the cold-banding technique used these methods. Kurabayashi (1952a, b) and Haga and Kurabayashi (1953) adopted this method for Japanese Trillium. Later, Fukuda and Kozuka (1958) collected all chromosome-banding data for Japanese diploid Trillium, and Fukuda and Channell (1975) and Fukuda and Grant (1980) developed the technique for Ameri- can and Canadian Trillium. Fukuda (1984) summarized the known data on chromosome banding.

Morphological analysis In the Himalayan mountain region T govanianum grows independently and the related species T tschonoskii and D. polyphylla are also found growing throughout the region. T tschonoskii is found growing on more lowland mountain ranges in Nepal, Bhutan, Sikkim, Yunnan, Japan and Sakhalin (Hara 1971, Fukuda and Kan 1966, Samejima and Samejima 1987, Uchino et al. 1994). D. polyphylla is isolated in more lowland situations in the Himalayas, Myanmar, Yunnan, Thailand, Vietnam and Taiwan (Hara 1969, Li 1998, Fukuda and Peng 1999). The morphological characters of T govanianum have features in common with T tschonoskii and D. polyphylla (Fig. 1). There- fore, a morphological comparison of these 3 species was pursued to determine their relationships. The floral organs of T govanianum are composed of 3 sepals, 3 petals, 6 (3 double) stamens and a 3-carpellate ovary (Fig. 2A). This is a typical Trillium floral composition as may be seen in the floral diagram of T tschonoskii (Fig. 2B). D. polyphylla has 5 different types within the species (Fig. 2C). One type shows 3 sepals, 3 petals but 7 stamens with a 3-carpellate ovary. Thus, the floral

Fig. 1. Habitat photographs of Trillium govanianum (A), Trillium tschonoskii (B) and Daiswa poly- phylla (C). 2001 The Origin of the Himalayan Trillium govanianum 107

Trillium govanianum Trillium tschonoskii

Daiswa polyphylla

Fig. 2. Floral diagrams of Trillium govanianum, Trilli- um tschonoskii and Daiswa polyphylla.

organ of D. polyphylla varies within a popula- tion. In contrast, the chromosome-banding component was the same for a population stud- ied in Nepal (Fukuda and Peng 1999). Fig. 3 shows the floral organs, ovary, stamen, petal and sepal in the 3 related species.

Ovary The ovary shapes are characteristic in each species. T govanianum has a dark purple Fig. 3. Comparative shapes of floral organs of Daiswa to a reddish purple ovary, whereas T polyphylla, Trillium govanianum and Trillium tschonoskii. tschonoskii has a white ovary. The fruit be- comes reddish in T govanianum and in D. polyphylla but in T tschonoskii it is a pale green.

Stamen The stamens of T govanianum and T tschonoskii have the same shape, but differ from those of D. polyphylla.

Petal The patals of the 3 species have a peculiar shape and differ in each species. T govanianum has a reddish purple linear-lanceolate, obtuse-base, acuminate apex petal. D. polyphylla has a yellowish long pole-like petal. T tschonoskii has a white oblong-ovate petal.

Sepal T govanianum has a sepal which is dark pinkish, linear-wide lanceolate, with an obtuse-base and an acuminate apex. D. polyphylla has a greenish sepal that is ovate-long lanceolate, and with an acuminate apex. T tschonoskii has a pail greenish sepal that is ovate-short oblong and with an acute apex. Fig. 4 shows the leaf shape of the related 3 species. Leaves: T govanianum has leaves that are 3 ovate, with acute apices, and rounded bases. T tschonoskii has leaves that are 3-wide rhomic-oval in shape with acuminate-apices and rounded bases. D. polyphylla has leaves that are 3-7 ovate-oblong with acute-apices, and rounded bases. 108 Ichiro Fukuda Cytologia 66

Daiswa polyphylla Trillium govanianum Trillium tschonoskii

Fig. 4. Comparative shapes of leaves in Daiswa polyphylla, Trillium govanianum and Trillium tschonoskii.

The rhizomes of T govanianum, T tschonoskii and D. polyphylla are all short and stout. It may be concluded that T govanianum belongs to the genus Trillium taxonomically, but with some differences from T tschonoskii. Some characters are in common with all species including Daiswa. To help further clar- ify the differences between these 3 related species chromosome information is required.

Chromosome analyses

By cold-induced banding methods, it has been found that each of the 20 chromosomes of Fig. 5. Drawing to illustrate the chromosome bands pre- sent in Trillium govanianum. T govanianum, exhibits peculiar heterochro- matic banding patterns, as may be seen in Fig. 5. By means of Feulgen staining, the chromosomes exhibited differential segments. The euchromat- ic portions are shown on the drawing in black and the heterochromatic areas are surrounded with a solid line. This banding pattern is stable within an individual and has the same pattern in all plants of T govanianum. The chromosomes are classified as A, B, C, D and E by arm length and cen- tromere position (Haga and Kurabayashi 1953, Fukuda and Kozuka 1958). T govanianum has 2 pairs of homologous chromosomes for each A, B, C, D and E letter. Chromosome A consists of Al and A2 in which there are 2 kinds of different banding patterns. Chromosome B consists of Bl—B4 but may be classified into 2 groups (B1, B2 and B3, B4). Chromosome C has 2 C 1 and 2 C2 chro- mosomes. Chromosome D has 2 D1 and 2 D2 chromosomes. Chromosome E has 2 El, an E2 and an E3 chromosome. From these data it may be shown that T govanianum is an allotetraploid com- posed of 2 genomes. In Fig. 6 a schematic diagram of all 20 chromosomes of T govanianum is given. Now the author tried to determine the origin of these T govanianum chromosomes, and hence, compared the T govanianum patterns with those of the related Trillium and Daiswa chromosomes patterns all of which have been analyzed by the same cold-induced banding method. Al has the heterochromatic band near the centromere. This same banding pattern was also 2001 The Origin of the Himalayan Trillium govanianum 109 found in T tschonoskii (Haga and Kurabayashi 1953, Fukuda and Kan 1966, Watanabe and Kayano 1971). A2 has a small heterochromatic part near the terminal end of the arm. Such a karyotype does not exist in other Trillium species, but Daiswa chromosome arms have the band at the subterminal location (Fukuda and Peng 1999). The B chromosomes of T govanianum were classified B 1, B2/B3, and B4. The inver- sion arm type of B 1, B2 was found in T cam- schatcense (Fukuda and Kozuka 1958). B3, B4 have a heterochromatic band at the subtermi- nal arm and Daiswa has such bands. The C 1 karyotype of T govanianum cor- responds with that of T tschonoskii, but C2 does not exist in T tschonoskii (Haga and Kurabayashi 1953, Fukuda and Kan 1966). C2 has a heterochromatic band at the subterminal Fig. 6. Diagrammatic illustration of the karyotype of arm that suggests it belongs to Daiswa. Trillium govanianum showing the banding patterns for The D1 karyotype of T govanianum cor- chromosomes A, B, C, D and E. The right-hand symbols responds with that of D. polyphylla (Fukuda show those bands in common with Trillium chromosomes and Peng 1999). D2 belongs to the Trillium (G) and Daiswa chromosomes (D). group because the same karyotype was found in T grandforum (Fukuda and Grant 1979). The El karyotype corresponds to D. polyphylla (Fukuda and Peng 1999) and E2, E3 belong to the Trillium group because the same karyotype was found in T ovatum (Fukuda 1969). As the result of the above chromosome analyses, it can be inferred that chromosomes of T go- vanianum comprise 2 genomes; one is the G genome derived from the Trillium group, very closely related to T tschonoskii. Another relationship is the D genome derived from the Daiswa group, which is very close to D. polyphylla. On Fig. 6, the letter G belongs to the G genome; D belongs to the D genome, respectively, in each chromosome at the right corner. From these chromosome data it can be concluded that T govanianum is an allotetraploid species that is the result of hybridization between the diploid T tschonoskii and the diploid D. polyphylla.

Discussion

By means of morphological and cytological analyses I have reached the decision that T gova- nianum is most likely a hybrid species between the GG genome of a plant in the genus Trillium and the DD genome of a plant from the genus Daiswa. However, some questions remain regarding the evolutionary development of this Himalayan Trillium hybrid species. The first question is why has such a natural hybridization become established between different genera? Usually, hybrids originate within a species or between species. The development of an in- tergeneric hybrid is rare as a result of reproductive isolation. What factors make possible poly- ploidization through intergeneric crossing in this Himalayan mountain region? On this point the most significant factor is the breeding system in both parental plants. The Asian Trillium species are predominantly outbreeders, whereas the American Trillium species have developed an inbreeding system (Fukuda 1990). Daiswa species have mainly an outbreeding system with vegetative repro- duction by means of rhizomes. Although we could not determine the pollination system of the orig- 110 Ichiro Fukuda Cytologia 66

inal T govanianum, it is suspected that the hybrid was developed through insects visiting the flow- ers as occurs in the Japanese Trillium species (Fukuda 1961). As shown in Fig. 2, the floral organs of D. polyphylla are variable. However, one type has the same features as that of Trillium. There- fore, a chance crossing by insect visitations between Trillium and Daiswa could give rise to the tetraploid species. Such a tetraploid species that possesses the double genome combination could lead to an individual having a wider habitat distribution (Fukuda 1967, for Achlys as an example). The second question concerning the Himalayan Trillium evolution is why has the diploid GG genome species disappeared from the Himalayas? On this problem we can hypothesize that the demise of the diploid species and the establishment of the tetraploid species were closely linked with each other. Namely, this dramatic event occurred as a result of climatic change in the Pleis- tocene age in Asia. Although the glacier was not covered in inland Asia, severe cold temperature and continued drought occurred in the Himalayas and Tibetan highlands areas (Vrba et al. 1995). Usually, Trillium plants grow under deciduous forests, and deciduous and coniferous mixed forests, with mesic humus. Trillium plants like a humid habitat because they increase by means of rhi- zomes. The intense sever cold and dry seasons took place 4 times in this region in the Pleistocene age and increased the desertification in the region. Many rhizomatous plants in the Liliaceae and the Trilliaceae perished and completely disappeared in that age. In such a critical period polyploidization was the only most effective method for their survival and perpetuation. Polyploid species can develop as they acquire a high degree of buffering against environmental change that takes place over long periods of time (Stebbins 1971, 1974). According- ly, although the diploid GG Trillium species deceased, tetraploid plants of T govanianum remained and perpetuated in the Himalayas. As another example, plants of tetraploid T tschonoskii also are distributed in the Himalayas, and their diploid species no longer exist there. In conclusion, through this research we can again hear and consider Stebbins' following sagac- ity, "Many individual polyploid genotypes have phenotypes which are able to tolerate a wide range of environmental conditions: they are general purpose genotypes" (Stebbins 1971, for the concept `general purpose genotype' see Baker 1965).

Acknowledgements Prof George Ledyard Stebbins Jr. died on January 19, 2000. I heard his inspiring lecture at the University of California, Berkeley, in 1962. I dedicate this manuscript to him. The photograph (Fig. 1A) was provided by the Bhutanese Botanist, Rebecca Pradhan. Prof William F. Grant, McGill University, Montreal, Canada made several helpful comments in reviewing the manuscript. I ac- knowledge these contributions gratefully.

References

Baker, H. G. 1965. Characteristics and Modes of Origin of Weeds. In: Baker and Stebbins (eds.). The Genetics of Coloniz- ing Species. Academic Press, New York. pp. 147-172. Chatterjee, A. 1973. Cytological investigation on certain genera of Liliaceae. Folia Biol. 21: 199-208. Darlington, C. D. and La Cour, L. F. 1938. Differencial reactivity of the chromosomes. Ann. Bot., N.S. 2: 615-625. ― and-1940 . Nucleic acid stervation of chromosomes of Trillium. J. Genet. 40:185-213. Fukuda,1.1961. On insects visiting Trillium flowers. Essays and Studies, Tokyo Wbman's Christian College 12:23-34.

-1967 .The biosystematics of Achlys. Taxon 16:308-316. - 1969 .Chromosome compositions of natural populations in Trillium ovatum. Science Reports, Tokyo Woman's Christian

College 7-11:110―137. - 1984 . Chromosome Banding and Biosystematics. In:Grant, W F.(ed.). Plant Biosystematics. Academic Press, Toronto .

pp.97-116. - 1990 . Breeding systems in American and Asian Trillium species by means of chromosome analysis. Plant Species Biolo-

gy 5:65―72. 2001 The Origin of the Himalayan Trillium govanianum 111

- and Channell, R. B. 1975. Distribution and evolutionary significance of chromosome variation in Trillium ovatum. Evo- lution 29: 257-266. - and Grant, W. E 1979. Chromosome compositions of natural populations in Trillium grandforum. Science Reports, Tokyo Woman's Christian Univ. 45-47: 509-547. - and - 1980. Chromosome variation and evolution in Trilliumgrandforum. Can. J. Genet. Cytol. 22: 81-91. - and Kan, E. 1966. A karyological report on the Trillium species of Ullung Island in Korea. Amatores Herbarii 27: 2-4. - and Kozuka, Y. 1958. Evolution and variation in Trillium. V. A list of chromosome compositions in natural populations of Trillium kamtschaticum Pall. J. Fac. Sci. Ser. V, Bot. VI 3: 273-319. - and Peng, C. T. 1999. Chromosome banding variation and evolution of Daiswa polyphylla. Proc. 29 Annu. Meet. Jpn. Soc. Plant Taxonomists: 64. Haga, T. and Kurabayashi, M. 1953. Genom and polyploidy in the genus Trillium. IV. Genom analysis by means of differen- tial reaction of chromosome segments to low temperature. Cytologia 18: 13-28. - and Watanabe, H. 1966. Chromosomes of a Himalayan Trillium in relation to the Japanese and American species. Proc. Jpn. Acad. 42: 160-164. Hara, H. 1969. Variation in Paris polyphylla Smith, with reference to other Asiatic species. J. Fac. Sci. Univ. Tokyo, Sect. 3, Bot. 16: 141-180. - 1971. The flora of eastern Himalaya, Second Report. Univ. of Tokyo Press, Tokyo. Li, H. (ed.) 1998. The Genus Paris (Trilliaceae). Science Press, Beijing. Mehra, P. N. and Sachdeva, S. K. 1976. Cytological observations on some W. Himalayan Monocots. II. Smilacaceae, Lili- aceae and Trilliaceae. Cytologia 41: 5-22. - and - 1977. Karyotype polymorphism in Trillium govanianum Wall. Cytologia 42: 331-338. Kurabayashi, M. 1952a. Differential reactivity of the specific chromosome segments in Paris. J. Fac. Sci. Hokkaido Univ. Ser. V, Bot. VI 2: 199-209. - 1952b. Differential reactivity of chromosomes in Trillium. J. Fac. Sci. Hokkaido Univ., Ser. V, Bot. VI 2: 233-247. Kurosawa, S. 1971. Cytological Studies on Some Eastern Himalayan Plants and Their Related Species. In: Hara, H. (ed.). The Flora of Eastern Himalaya. Second Report. Univ. of Tokyo Press, Tokyo. pp. 356-364. Kumar, V. and Subramaniam, B. 1989. Chromosome Atlas of Flowering Plants of the Indian Subcontinent. Vol. II. Mono- cotyledons and Bibliography. Botanical Survey of India, Calcutta. Samejima, K. and Samejima, J. 1987. Trillium Genus Illustrated. Hokkaido Univ. Press, Sapporo. Stebbins, G. L. 1971. Chromosomal Evolution in Higher Plant. Edward Arnold Ltd., London. - 1974. Flowering Plants-Evolution above the Species Level. Harvard Univ. Press, Massachusetts. Uchino, A., Takayama, M., Li, H. and Gu, Z. 1994. Distribution and circumstances of the habitat of Trillium tschonoskii in Yunnan Province of China. Memories. Fac. Gen. Edu. Kumamoto Univ. Nat. Sci. 29: 61-73. Vrba, E. S., Denton, G. H., Partridge, T. C. and Burckle, L. H. (eds.). 1995. Paleoclimate and Evolution. Yale Univ. Press, New Haven. Watanabe, H. and Kayano, H. 1971. Karyotype analysis of natural populations of Trillium tschonoskii. Jpn. J. Genet. 46: 231-234.