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1. Germplasm Resources

Germplasm resource is a collective term for all genetic resources. Plant germplasm resource is a precious treasure for the human being. There is no way to gain excellent cultivars without good germplasm resources.

China owns varieties of tree peony germplasm resources. Protection, preservation, collection, study and use of these germplasm resources are the most urgent and important tasks.

1.1 The Origin and Evolution of Wild Species

1.1.1 The Taxnomic Status of the Genus Paeonia (Paeoniaceae)

Paeonia is Extremely Isolated in the Systematic Classification In 1735, Carolus Linnaeus constructed the genus Paeonia based on a perennial herbaceous plant native to the Balkan Peninsula, Europe. British botanist H.C. Andrews released the formal name Paeonia suffruticosa Andr. in 1804 depending on the cultivar introduced from China. For long time, the taxonomical systems, such as Engler, Bentham and Hooker et al., which all put the genus Paeonia into Ranunculaceae, have been widely used in the world until someone thrown doubt upon them at the beginning of 20^th century. British botanist W.C. Worsdell suggested setting up Paeoniaceae by separating Paeonia from Ranunculaceae based on the significant differences in anatomy between Paeonia and other genera of Ranunculaceae.

During the 1950s, some researchers found a lot of important evidence from anatomy, the feature of flower, the structure of ovule, the development of stamen, the characteristics of chromosome and cytogenetics, embryogenesis, the modality of pollen, the structure of ektexine, and the chemical components, which are enough to prove that Paeonia is different from other genus in Ranunculaceae. After that, an independent family Paeoniaceae is commonly accepted, however, the position of Paeoniaceae in a systematic classification became the argumentative focus. Most popular systems had upgraded Paeoniaceae to Paeoniales in the 1980s and consequently brought about argument in academe about the position of Paeoniales in a systematic classification. Paeoniaceae was respectively put under Ranunculanae, Theiflorae, Annonanae and other corresponding grades by the different scholars and there is no consistent viewpoint so far (Pan K.Y. ,1995; Peng Z.H. et al., 2000). It is out-of-the-way in plant systemic taxonomy that there are so many different opinions about the taxonomic positions of both Paeoniaceae and Paeoniales. It reflects the speciality of Paeonia (Paeoniaceae) in a systematic classification. Paeonia is a quite independent taxon and its close relative remains unknown.

Paeonia is the Most Primitive Angiosperm

Paeonia (Paeoniaceae) is considered one of 60 most primitive genera (families) in academe. It has acentric development stamens, perigynous disc, thick carpel, wide stigma and the aril stemmed from placenta. Paeonia has large chromosome and the basic number is 5 («=5). Free nuclei stage appears in the early stage of embryogenesis and it has special chemical components -paeoniflorin, paeonol, etc. The study of embryology began in the 1950s and the advances of reproductive biology in recent years not only provided more and more evidence to its speciality in an-giosperm and similar characteristics of ferns, also indicated that a clear genetic and evolutional ancestral characteristics come down from ferns (Peng Z.H. et al. 2000):

(1) The tapetum of Paeonia anther derived from the primary sporogenous cell and it is homologous with pollen mother cell (Cheng F.Y., 1996). Initiation of the tapetum of Paeonia anther is almost the same as that of ferns.

(2) Paeonia is thick ovule and the nucellar cells consist of marginal and sporogenous cells. Only the sporogenous cell located in chalaza extremity has been transformed into megaspore mother cell. Homologous origin of the nucellar cell and the megaspore mother cell is a special development stage of Paeonia.

(3) Paeonia can produce many megaspore mother cells and embryo sacs in one nucellus. Many megagametophytes develop in one ovule simultaneously is a unique common characteristic of Paeonia in angiosperms. This phenomenon merely happens in a certain species of Selaginella and Isoetes.

(4) In the procedure of embryogenesis, Paeonia /ygote becomes a series of free nuclei instead of cell wall after the first division. Except that the free nuclei proembryo cell doesn' t directly generate embryo, it is almost the same as Cycas and Ginkgo in initial gymnosperms. It is considered as either the result of parallel evolution of Paeonia and gymnosperm or the result of genetic expression of the ancestral characteristic that the development of seed or fern

Chinese Tree Peony (Xibei, Xinan, Jiangnan Volume) megagametophyte' s gets through the free nuclei stage.

(5) Paeonia has a special feature of embryo dormancy including epicotyl and hypocotyl dormancies, and epicotyl dormancy is deeper than hypocotyl. The suitable procedure and temperature required to break two types of dormancies are different. The seeds should be harvested when close to mature and should be stored in the moisture places. It is obviously a kind of remained ancestral characteristic, namely, their ancestor must has experienced the wet weather of the tropics and subtropics. In addition, Paeonia has a special character of self-reproduction. The wild tree peony species can be divided into two groups by their reproductive features. One is mainly vegetative reproduction and partly seed reproduction, the other is sexual reproduction completely by seed (Cheng F.Y., Li JJ. et al., 1995). The osculant group with sex differentiation which intermediates asexual and sexual reproduction is more primitive in tree peonies. From this point, the reproduction property of peony is almost the same as that of fern.

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China is the Original Phylogenetic Center of Paeonia

The genus Paeonia is divided into three sections including Moutan, Onaepia and Paeonia (Stern, 1946). According to the speculation of character evolutionary trend (Pan K.Y., 1995), the tree peony, Moutan, is most original followed by Onaepia, while Section Paeonia is younger and more evolutionary. The plants of section Moutan are the diploid subshrub with numerous flowers of five carpels. These botanical characteristics are all original. Sect. Moutan contains 9 wild species and all of them are native to China. Sect. Onaepia combines both original characters of numerous flowers and diploid etc. and the specialized characters with herbaceous habit, fusiform tuberized root and narrow elliptic leaflets. In this section, only two species are native in western North America. Sect. Paeonia contains 22 species and some original herbaceous species also have been differentiating in China. These species share the same distributional areas with Sect. Moutan. Sect. Paeonia has drifted towards west and has been dramatically differentiated into 12 species in the Mediterranean basin and most of which are tetraploid. The eastern China, the southeastern Qingzang Plateau, the mountains of Qinling and Bashan and the Loess Plateau should be the centers of origin, evolution, development, as well as diversity of the genus Paeonia in the world.

1.1.2 The Speculation of the Origin of Paeonia

Somebody thought the genus Paeonia originated in the Cretaceous period, about a hundred million years ago, when angiosperm thrived. As for the phylogenesis of Paeonia, the differentiation and formation of Sect. Onaepia was earlier than Sect. Paeonia. According to the speculation of Stebbins and Ellerton (1939), two species of Sect. Onaepia probably originated in the early Tertiary or the late Cretaceous period. Recently, the researchers considered this differentiation happened latest in the Miocene, at least 13 million years ago, because the latitudes of their distribution are almost the same as those of Liriodendron. According to the ancient weather of the "Bering Bridge", L. chinense in East Asia and L. tulipifera in North America must have been linked with each other before 12 million years (Wolfe, 1972; Hong, 1983). The results of the alloenzyme analysis and the restriction fragment length polymorphism analysis of cpDNA suggested that the differentiation of these two species occurred 13 million years ago (Hong, 1993; Parks et al., 1990).

However, some researcher thought it hard to explain these two species of Onaepia far away from East Asia by the European route or diffusing to the west coast of North America through the "Bering Bridge". For the wild populations of the genus Paeonia, the fertility is generally poor, seed is larger and the germination rate will decrease after drying and insolating. On the other hand, the mature of seed is in August when the foods of birds and beasts are rich. Therefore, the long distance diffusion of population through seed is impossible by animals and rivers. The eastern side of the East Asian Continent and the western side of the North America were linked with each other in the Pangaea Paleozoic and then separated because of the Plate Tectonics Activity. From this point, the herbaceous peonies should occur in the stage of linked ancient continent. They drifted away with the continents as the linked continents broke in the late Trias and consequently formed discontinuous distribution pattern - East Asia and North America which were separated by the Pacific Ocean. Additionally, according to the characteristics of the genus Paeonia phylogenesis, combining with the ancient geography and weather's changes, the origin of Paeonia can be dated up to the Carboniferous of the Paleozoic. The differentiation and development happened in the Permian. The original herbaceous peonies appeared in the Trias in accordance with the formation time of the Pacific Ocean resulted from the plate drift. Modern Paeonia thrived from the Jurassic to the Early Cretaceous period. Strong selection pressures of long time dryness and increased temperature are the main reasons of transformation of the tree peonies to the herbaceous peonies (Peng Z.H. et al., 2000).

1.1.3 The Relative Relationships and Character Evolutionary Trends of Section Moutan in Paeonia

The Relative Relationship Analysis of Various Taxa

Some advances have been made in the studies of relative relationships in the wild species of section Moutan of Paeonia. Based on the morphological analysis, some researchers thought P. qiui and P. jishanensis are close relatives, other studies indicated that P. ostii, P. rockii and P. jishanensis are close relatives too. However, different studies resulted in some differences in conclusion. For example, Yuan T. et al. (1999) used the data of pollen conformation and external characters of plant to analyze the classification relationships between 5 species of Subsection Vaginatae and P. yananensis by the UPGMA cluster analysis method. She claimed that P. qiui and P. jishanensis have the closest relationship, the next is P. ostii and P. rockii. P. decomposita has a far relationship with the four species above. The character of P. yananensis is between P. jishanensis and P. rockii but closer to P. jishanensis, therefore it can be considered a sub-taxon of P. jishanensis. However, Hong D.Y. etal. (1999) took P. yananensis as a hybrid of P. jishanensis and P. rockii. Yu L. et al. (1998) made a quantitative similarity analysis of leaf protein of 6 wild species of tree peony and concluded that there is a close relative relationship between P. rockii, P. decomposita, P. jishanensis, P. lutea and P. potaninii, but it is not that case for P. ludlowii. Zou Y.P. et al. (1999a) conducted systematical study of Section Moutan using RAPD molecular marker. 59 specimens of 15 populations of 7 species (including P. delavayi and P. lutea of Dian Moutan) were covered in this study. She constructed a dendrogram of 121 polymorphism loci from 10 primers using UPGMA cluster method and discussed the intraspecific and interspecific genetic relation-

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ship in Section Moutan. As a result, the species of the two Subsections were divided into two main branches, but the relationship between the species of Subsection Vaginatae was displayed differently in the result of this study. In the cluster dendrogram, P. qiui and P. ostii converged first and then P. rockii, P. decomposita and P. jishanensis joined in turn, finally one big branch formed. T. Sang et al. (1995) found the reticulate evolutionary feature of Paeonia by the sequence analysis of the internal transcribed spacers (ITS) of nuclear ribosomal DNA (nrDNA) in 33 species of the genus Paeonia. He constructed an interspecific phylogenetic tree in 12 species which obviously did not derive from hybridization by the parsimony analysis. Later, he (1997a) investigated the phylogenetic relationships, reticulate evolution and biogeography of 32 Paeonia species (P. jishanensis and P. delavayi included) using the coding region of the matK gene and two intergenic spacers, psbA-trnH and frnL(UAA)-fmF(GAA) of cpDNA, and made the clearest gene tree in the studied species. He put forward a refined hypothesis of the species phylogeny of Section Paeonia by comparing this gene tree with the phylogenetic tree made by using ITS sequences. T. Sang et al. (1997b) and Ferguson et al. (2001) thought it's better to use gene ADH to build the interspecific relationship of Paeonia than to use ITS and chloroplast gene matK. They also successfully proved that P. officinalis, a tetrap-loid hybrid, derived from two heterogenous tetraploid species. Unfortunately, the interspecific relationship of Section Moutan was not yet resolved by this method. B.C. Tank and T. Sang (2001), using the nuclear-encoded chloroplast-expressed glyc-erol-3-phosphate acyltransferase (GPAT) gene, investigated the phylogenetic relationship of 13 Paeonia species (19 accessions) including P. rockii, P. decomposita, P. jishanensis and P. delavayi. For the first three species, there are obvious differences between the result of this research and those of Zou Y.P. (1999) and Yuan T. (1999). Recently, Zhou Z.Q. et al. (2003) did the phylogenetic analysis based on the morphologic evidence of 40 populations of all the wild tree peony species. The phylogenetic relationship of Section Moutan was proposed. The result is supported by molecular systematics and it suggested P. jishanensis and P. qiui have the closest relationship followed by P. suffruticosa, P. ostii, P. rockii, P. decomposita, P. delavayi (P. lutea and P. potaninii included) and P. ludlowii. In the author' s opinion, in order to obtain the results in accordance with the evolutional facts, the further studies of the molecular systematics and other related subjects integrating the analysis of the character evolution trends are necessary.

Advances in Evolutionary Trends in Characters

Some speculations over character evolutionary trends in Paeonia were proposed (Pan K.Y., 1995; Li J.J., 1999) and advances of importance were made in recent years (Lian Y.S., Zhao M.G., 2002; YuanT. etal.,2003).

Evolutionary Trends in Carpels The structure of carpels of the genus Paeonia Section Moutan is primitive. In order to better adapt to insect pollination and enhance the resistance to cold and dry weather, the carpels expressed the following evolutionary trends.

—Carpels become thick pubescent from glabrous ones (except for Subsection Delavayanae and P. decomposita with thin flosses only in the dorsal suture and the ventral canal).

—The stigma curling extent and the density of papilla are from low to high, which benefits to increase the pollination area.

—Carpels vascular tissue gradually enhance, vein pit becomes clear from unclear.

—The stigma tends to be longer from the shorter to make the differentiation of carpels more complete.

—The cicatrization extent of ventral suture of carpels is from low to high, the mechanical tissue increases except vascular tissue of ovary wall, so the capacity of protecting ovules and seeds is" enhanced. The carpel structure of species of Subsection Delavayanae is more primitive (especially in P. delavayi and P. lutea). As a comparison, the Subsection Vaginatae is more developed and P. decomposita is between the two.

The Origin and Evolutionary Trends in Discs Disc is an important character of classification of subsection, species or subspecies in Paeonia (Stern, 1946). Since 1960, the author has been using "Fang Yi (Ovary-Enveloping Coat)" as the name of the floral disc of tree and herbaceous peony. It was recently supported by Zhao M.G. (2002) in terms of the conformational and anatomic characters:

—The disc does not originate from receptacle tissue protuberance but a part of tissue adnascent in the back of carpel. It is an accessorial organ which is secondarily differentiated from the same carpel primordium. Each carpel has the corresponding disc.

—Carpel always possesses disc or its variation structure and remained vestige, whether it normally developed or not. In the simple flower, discs form the cup or plate shape which possibly resulted from the secondary concrescence between the discs of each carpel.

—Mature leathery disc is obviously different from the fleshy disc in conformation character and anatomic structure. The former has developed tissue, the most part of which surrounding carpel is thin. Many dense canaliculi can be seen in the transverse section. The tissue of the latter is less developed. But in the primary development stage, leathery disc is similar to fleshy disc and leathery trends are found in some taxa of the subsection Delavayanae. It indicated that they have the same origin, the former comes from the latter. Leathery disc gradually formed as plants diffuse towards the cold and dry areas.

—Developed vagina appears in the genus Paeonia at the primary stage of development. It suggested that the disc of this genus may stem from vagina and the carpel from leaf. Namely, as a whole, carpel and disc are transformed from a complete leaf. According to the discussion above, in the Section Moutan of genus Paeonia, Subsection Vaginatae develops from the relatively primitive Subsection Delavayanae.

Other Evolutionary Trends in Characters Evolutionary Trends in Petal In the Section Moutan of genus Paeonia, the petal of primitive taxa spirally arranges and its apex is small gibbose needle or sunken. The petal of developed taxa consists of two annulus with deep sunken apex to form two rips. The evolutionary trend is from small needle on apex (the Subsection Delavayanae) to deep divided rips (the Subsection Vaginatae). Evolutionary Trends in the Pollen Grain Exines The basic evolutionary rale in the pollen grain exines is: small foveolate —> foveolate^ reticulate—* coarse reticulate. The pollen grains with rugate undulant and other intermediary patterns are found recently. Both reticulate and rugate undulant patterns are isochronous in morphology evolution of pollen grain.

Evolutionary Trends in Leaf Shape and Flower Color In the Subsection Vaginatae, leaflets follow the following evolutionary trends - from few to numerous for the number, from wide to narrow

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Figure 1-1 Paeonia ludlowii

size and from accrete to free type, however, the basic trifoliate structure still remains. Pink is the most primitive color of flower. White and pinkish flower should develop later. At the base of petal, the color development from pale purple faint to small purple blotch and then to obvious big purple blotch, is a possible evolutionary route.

1.2 The Status of Wild Germplasm Resources and Their Protection

1.2.1 The Characters of Wild Species and its Varieties

There are about 9 wild species, all native to China, in the genus Paeonia Section Moutan. The recent studies indicated that these species have a rich genetic diversity. As described above, F.C. Stern divided Section Moutan into two subsections with obvious differences in both morphological characteristics and ecological habits. These two subsections also have different practical prospects in breeding.

The Subsection Delavayanae

Four species are involved in this subsection. They are distributed in Southwest Sichuan, Southeast Tibet, Middle and North Yunnan and West Guizhou.

Paeonia ludlowii (Stern & Taylor) Hong, [ P. ludlowii (Stern & Taylor) JJ. Li et D. Z. Chen; P. lutea Delavay ex Franch. var. ludlowii Stern et Taylor]

A large deciduous shrub up to 3.5 m tall. Thick fleshy roots attenuated downward to the tip but no expanded ellipsoid. Bark gray-brown and exfoliated. Leaves big, biternate, pinnate, compound. Leaflets 9, glabrous, above green and below gray, almost sessile, mostly 3-partite, then 2-teethed, acuminate at the apex. Flowers 3 or 4 per shoot, terminal and axillary, diam. 10-12cm.

Bracts 4 or 5. Sepals 3 or 4, arranged by size. Petals spreading, pure yellow, occasionally white, obovate, apex rounded. Filaments yellow. Disc fleshy, yellow, papillary. Carpel(s) 1 or 2, glabrous. Stigmas yellow. Follicles cylindric. Seeds big and globose. Flowering late May to early June. Fruit August (Fig. 1-1).

P. ludlowii is distributed in a quite narrow area. The wild species is only found in the Brahmaputra Gorges at altitudes between 3,000 - 3,700 m in SE Tibet. The plants are tall and have large leaves and beautiful flowers (Fig. 1-2). Ludlow and Sherriff brought P. ludlowii to England from Tibet in 1936 and introduced it many times later. Until 1953 P. lutea var. ludlowii was published (Bot. Mag., London, 1953, t. 209). Nowadays, P. ludlowii, as a substitute of P. lutea, has been widely cultivated in Europe and America and used as parent plants in breeding. The resource investigation of this species was done late in China. After investigation from April to May in 1986, Zhang Q.X., from Beijing Forestry University, found a 20 hm² sized population (pop.) in Milin of 3,100 m in altitude on the south coast of Brahmaputra where P. ludlowii is a dominant species. He also found that this species has only 1 or 2 carpel(s) and blooms 2 or 3 weeks later than P. lutea. Since then, Chen D.Z. et al. of Yuzhong Peace Peony Garden in Gansu investigated and introduced it respectively in 1991 and 1993. Based on the field investigation and the observation of introduced plants, Li JJ. et al. found obvious differences between P. ludlowii and P. lutea in both morphological and ecological characters and took it as a species (Li JJ. et al., 1995). The analyses of karyotype and leave protein also indicated that it was obviously different from P. lutea. Therefore, the classification position of P. ludlowii should be established (Li JJ. etal, 1998). During this period, Hong D.Y. etal also performed the investigation and reported the same viewpoint.

P. ludlowii is used as not only an ornamental plant in the suitable areas but also an important breeding material to produce larger yellow peonies. In addition, its root cortex is used as "Dan Pi" in traditional Chinese medicine.

P. delavayi Franchet, Bull.

Deciduous subshrub, to 1.5m tall. Glabrous throughout. Roots tuberous, expanded ellipsoid. Annual stems herbaceous, blackish red-purple. Scales at the base of branchlet. Proximal leaves 2-ternate, pinnate segmented, compound. Segments lanceolate to oblong-orbicular lanceolate, margin entire or occasionally dentate, leave back gray. Flowers 2-5 per shoot, usually 3, terminal and axillary, diam. 6-8 cm. Petals 9-12, red to purple-red, base darker, glossy. Stamens numerous, 0.8-1.2 cm long; filaments, dark purple. Disc fleshy, wholly enveloping the base of carpels, apex segments triangular or ovate-orbicular. Carpel(s) 2-5, glabrous. Stigmas purple. Flowering in early May. Fruits July to August. 5-7 black seeds per follicle. Usually persistent large involucres (Fig. 1-3).

P. delavayi is mainly distributed in NW Yunnan, SE Sichuan and SE Tibet (only in Zhanang). It grows in thickets, sparse forests, and

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Figure 1-2 P. ludlowii 's ecological environment (Zhilin, Tibet)

Figure 1-3 P. delavayi

Figure 1-4 (left) one of P. delavayi's ecological environment

Figure 1-5 (Middle) one of P. delavayi's ecological environment

Figure 1-6 (right) one of P. delavayi's ecological environment (Lijiang in Yunnan)

the conifer forests of the southern slopes at the altitudes of 2,300 - 3,700 m (Fig. 1-4, Fig. 1-5, Fig. 1-6). The holotype of P. delavayi was collected by A. Delavay in Lijiang, Yunnan in 1884. This species was reported by A.R. Franchet in 1886. The plant has strong stems with beautiful flowers, thus it is a wonderful ornamental plant as well as a good germplasm in breeding. American A.P. Saunders successfully produced the new cultivars with dark red-purple to dark maroon flowers by crossbreeding between P. delavayi and Japanese tree peony cultivars. The root bark of P. delavayi is used as a medicine material of "Dan Pi", too.

P. delavayi was investigated and introduced by Li J.J., He L.X. and Chen D.Z. et al. during 1998 -2001. The plants introduced from Lijiang and Zhongdian, Yunnan to Lanzhou could normally grow, bloom and fruit. In the fall of 2001, He L. X. observed that some plants bloomed in the second time from late September to October in gardens. If it is protected by a big shed in winter, all buds of the biennial and triennial branches can become flower buds and normally bloom. In the spring of 2002, 65 flowers were bloomed in the biggest plant. It indicated the good characters of flower formation in this species.

P. potaninii Kom. (P. delavayi Franch. var. angustiloba Rehder & E.H. Wilson)

Deciduous shrub, 1.0-1.5 m tall. Stems orbicular, pale green and glabrous. Roots tuberous and ellipsoid expanded. Proximal leaves 2-ternate, compound, 2-segments, 3-5 or much-partite segments, narrow-lanceolate, 0.5-1.0 cm. Flowers red to red-purple, diam. 5-6 cm; petals 9 -12. Bracts and sepals 5-7. Stamens numerous, filaments red. Carpels 2 - 3, glabrous. Stigma thin and curly. Disc fleshy, 2-3 mm high. Flowering May. Fruit August (Fig. 1-7).

It is found in Yajiang and Batang of W Sichuan, Songming, Lijiang and Zhongdian of NW Yunnan, alt. 2,300 - 3,700 m (Fig. 1-8). This species was introduced to England by Wilson in 1904 and first bloomed in Veitch' s Coonbe Wood Nursery in 1991. The flower is small but the leaves are slim and elegant. Therefore, it is a good parent plant to breed a novel cultivar with wonderful leaf shape.

P. lutea Delavay ex Franch. [ P. delavayi Franch. var. lutea (Delavay ex Franch.) Finet & Gagnep.]

Deciduous shrub, 0.5 - 1.5 m tall. Stems orbicular, gray and glabrous. Roots tuberous, ellipsoid expanded. Annual branches red-purple. The coat of branches over biennial, desquamated pieced. Proximal leaves 2-ternate, compound, 2-segments 3-5

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Figure 1-7 P. potaninii

Figure 1-9 P. lutea

Figure 1-8 P. potaninii's Ecological Environment (Yajiang, Sichuan)

Figure 1-10 P. lutea 's ecological environment (Zhongdian, Yunnan)

partite. Segments lanceolate, at least 1.0 cm wide. Flowers 2-3 occasionally single, at the apex and axillary upside. Petals yellow to yellow-green, occasionally brown blotch at base. Stamens numerous. Filaments yellow. Disc fleshy, 3-5 mm high, yellow and dentate. Carpels 3-6 usually 5. Flowering May. Fruits August (Fig. 1-9).

P. lutea is the widest ranged species in the Subsection Delavayanae. It is distributed from Jingdong of Yunnan to Mt. West of Kunming and Mt. Liangwang (alt. 1,860 m) and extends to NW Yunnan, SW Sichuan, W Guizhou and SE Tibet. A lot of varieties under species are found in these areas. This species has a rich variation in the leaf shapes and a strong adaptability to all sorts of ecological environments (Fig. 1-10, 1-11).

Somebody has divided this species into several variations. According to the author's investigation and observation of many years, the following five varieties have their different practical values in breeding.

P. lutea var. lutea

P. lutea var. brunnea JJ. Li This variety is about 1.5 m tall. There are big brown blotches at the base of petals. It is found in the mountainous thickets in alt. 2,300m in Lijiang, Ludian, Huadianba of Cangshan, Yunnan. It was introduced to the Peace Peony Garden in Lanzhou, Gansu(Fig.l-12).

P. lutea var. humilis JJ. Li et D.Z.Chen This variety is only 0.5 tall. It is found in the glades and thickets in alt. 2,200 m in Hala villa; of Zhongdian, Yunnan. It was introduced to the Peace Peony Garden Lanzhou, Gansu(Fig .1-13).

P. lutea var. trollioides (Stapf et Stern) J.J. Li (P. potanir var. trollioides Stapt et Stern) The plants of this variety are almc erect with many flowers like the genus Trollius (Ranunculaceae Forrest introduced it to Europe from Yunnan in 1914. It is cultivati in England(Fig. 1-14).

P. lutea var. alba J.J. Li (P. potaninii f. alba) The flowers of this variety are white. It is found in the riv beaches of northern Weixi, Yunnan. The population with pa white flowers is heavy fragrant(Fig.l-15). A series of intermedia types, which came from interspecific hybrids of P. delavayi and lutea(Fig.l-16), have been found in Tangdui and Hala villag Zhongdian, Yunnan and other places. In addition,Sun J.Z. found yellow-flowered peony with characters between the tree at herbaceous peony in Chuxiong, Yunnan(Fig.l-17). During 199' 1998,Li JJ.,He L.X. and Chen D.Z. et al. investigated SW Sichu: and NW Yunnan for many times and successively introduced tl plants from six populations of P. lutea. These plants all not on normally grew, flowered and born fruits in Lanzhou, their se« offspring also successfully bloomed, fruited and retained identic

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Figure 1-11 P. lutea 's ecological environment (Xishan, Kunming, Yunnan)

Figure 1-12 P. lutea var. brunnea J.J. Li

Figure 1-13 P. lutea var. humilis J. J. Li et D.Z. Chen

Figure 1-14 P. lutea var. trollioides

characteristics of the original species. Secondary flowering has been found in the introduced plants in other introduced places. In early time, P. lutea was introduced to Europe and America and used as parent plant to generate novel cultivars. P. x lemoinei Rehd. was successfully bred by cross-breeding between P. lutea and P. suffruticosa. French Louis Henry conducted some hybridization experiments in the Museum of Paris and then in Leimuwana Nursery Corporation. After that, Professor A.P. Saunders continued to do this research and bred a series of beautiful hybrids in America.

The Subsection Vaginatae

There are five species in this subsection. They are distributed in NW Sichuan to Middle South Gansu along mountains of Qinling and Bashan extended east to West Henan, West Hubei, NW Hunan, SW Anhui, and north to the forest of the Loess Plateaus.

P. decomposita Hand. -Mazz. (P. szechunica Fang) Deciduous shrub, glabrous throughout. Stems 0.7-1.5 m tall. Bark gray-black, peeling off in exfoliate. Branches columniform. Squamae persistent at the base of stems. Proximal leaves divided 3 - 4 times compound. Leaflets (29-) 33-63; terminal leaflets ovate to obovate, 3-partite to middle or almost partite entire, segments again 3-lobed; lateral leaflets olivary or rhombic-olivary, 3-lobed or not lobed but coarse teethed. Flowers single, solitary terminal, diam. 10 - 15cm. Bracts 3-5, linear-lanceolate. Sepals 3-5, obovate, apex

mucronate. Petals 9 - 12, rose to pink, obovate; apex irregularly incised or calathiform. Stamens 1.2 cm long. Filaments white. Disc leathery and cotyloid, 1/2 - 2/3 enveloping carpels, apex segmented triangular. Carpels 4 - 6, subulate. Styles vertical; stigma flat and convolved counterclockwise. Flowering late April to early June. Fruit August.

This species has been divided by Mt. Qionglai into two allopatric subspecies in Minjiang drainage area and Jinchuan-Daduhe drainage area.

P. decomposita subsp. decomposita

The leaflets are ovate or obovate, partite (Fig. 1-18). It is distributed in the regions of Ma' erkang, Jinchuan, Danba, Kangding of Sichuan Province (Fig. 1-19). It is also found in South Gansu. This species is quite common in the thickets of Daduhe drainage area (alt. 2,050-3,100m) in Jinchuan.

P. decomposita subsp. rotundiloba D.Y. Hong

The number of carpels in this subspecies is 3 to 4. Leaflets are ovate-orbicular. Segments of leaves are oblong with round or cuspate margin. It is quite commonly found in thickets, secondary forests and needle-leaved forests in alt. 2,100-3,100 m of Wenchuan, Maoxian, Heishui, South Songpan and Li Xian in Minjiang river drainage area of Sichuan Province.

There is no report that P. decomposita is used in breeding. Only a few were cultivated in NW Sichuan. The plant is tall, but stems and leaves are slim. Flowers are delicate and charming at the

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Figure 1-15 (left up) P. lutea var. alba J. J. Li

Figure 1-16 (left down) A series of intermediate types hybrids of P.delavayi and P. lutea

Figure 1-17 (right) The roots tuberous ofP.delavayi and P.lutea

Figure 1-18 (below left)P. decomposita

Figure 1-19 (below right) P. decomposita 's Ecological Environment (Maerkang, Sichuan)

beginning of blossom. It should be used as an ornamental plant as well as breeding material in the near future.

P. qiui Y.L. Pei et D.Y. Hong

A deciduous shrub, 0.6-0.8m tall. Branch barks brown-gray, longitudinally striate. Roots tuberous, turions present. Proximal leaves 2-ternate compound; leaflets 9, often purple-red suffused above, light green below, ovate or ovate-orbicular, apex obtuse or acute, base rounded, mostly margin entire. Flowers single, solitary, terminal, 8 -12 cm cross. Petals 5-9, pink or pale pink, spreading; stamens 80-120; filaments pink to pale pink. Styles very short; stigma flat and anticlockwise curling like lobe, most purplish red, circumvolve 90 - 360 degrees, half clockwise and contraclockwise. Disc pale red-purple, leathery, wholly enveloping carpels; carpels 5, densely tomentose white or light yellow. Follicles splindly, densely

yellow wirehaired. Seeds ovoid, black and glossy. Flowering late April to late May, fruit July to August (Fig.1-20).

It is narrowly distributed and only found mostly in cliffs or steep slopes in alt. 1,650 - 2,010 m in Songbai town of Shennongjia, Hubei (Fig.1-21). This species is common found in the hilly country of Baokang close to Shennongjia. In addition, it is also located in Xixia County of SW Henan. Li J.J., He L. X. and Chen D.Z. et al. have investigated P. qiui for several times in Baokang, Hubei and introduced it to Lanzhou where this species can normally grow, bloom and bear fruits.

The plant of this species is short. The beautiful flowers are pink in the beginning of blossoming. P. qiui was first published by Pei Y.L. and Hong D.Y. in 1995. Although this species had little effect on modern cultivars of tree peony, the features of shorter stems and red-purple fainted leaves are important germplasm resources to breed new cultivars with low height and special leaf color.

P. jishanensis T. Hong et W.Z. Zhao (P.suffruticosa subsp. spontanea (Rehder) S.G. Haw et L.A. Lauener]

Deciduous shrub, 0.5 - 1.5 m tall. Stem barks brown-gray, longitudinally striate. Roots tuberous.

Proximal leaves 2-ternate compound; leaflets 9 or more, nearly round or ovate-orbicular, 1-5 segmented, segments coarsely toothed. Leaves villous below, lateral leaflets nearly sessile with coma at the base. Flowers solitary, terminal. Bracts 3 or 4, sepals 3. Petals 6-8 (10), white, occasionally pink or light purplish red at the base. Stamens nemerous. Filaments pale purplish red, white near top. Disc, pale purplish red, apex dentate. Carpels 5, densely yellow-white coarsely tomentose. Stigmas dark purplish red. Young fruits thickly gray-white rough tomentose. Seeds black and glossy. Flowering late April to early May. Fruits August (Fig. 1-22).

This species is naturally distributed in thickets and secondary deciduous broad-leaved forest in alt. 900-1,700 m in Jishan and Yongji of Shanxi, Jiyuan of Henan, Huashan, Tongchuan and Yan' an etc. of Shaanxi.

It was regarded as a wild species which is a close relative of

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Figure 1-20 P. qiui

Figure 1-21 P. qiui and its ecological environment (Baokang, Hubei)

Figure 1-22 P. jishanensis

cultivated tree peonies (Haw & Lauener 1990; Pan 1979; Stern 1946) or as one of its closely related ancestors (Li J.J. 1987; 1999). However, since P. jishanensis is propagated mainly via clone and there are obvious differences in both leaves with or without villi and the morphologic features of leaflets and segments between P. jishanensis and cultivated tree peonies, Hong D.Y. (1998) considered that a single plant with light pinkish purple single flower, which was said to be introduced from the hill to Secaogou, Shigunping village of Muzhijie, Songxian County, Henan Province and a wild tree peony found in cliff of Yinpingshan, Chaohu, Anhui Province were the wild relatives of modern cultivated tree peonies. He also published a new subspecies - P. suffruticosa Andrews subsp. yinpingmudan Hong, K.Y. Pan et Z.W. Xie, however whether this subspecies is accepted is still under discussion.

P. ostii T. Hong et J.X. Zhang

Deciduous shrub about 1.5 m tall. Stem barks brown-gray, longitudinally striate. Proximal leaves 2-pinnate compound; leafletslS, ovate-lanceolate or narrow oblong-ovoid, apex gradually acuminate, base cuniform or orbicular or nearly truncate, margin entire. Terminal leaflets occasionally 1-3 lobed, above costa near base rough tomentose. Lateral leaflets nearly sessile. Rowers solitary, terminal, single, diam. 12.5-13 cm. Bracts 3, ovate-orbicular or narrow oblong-ovoid. Sepals 3, broadly ovate-orbicular, apex acute caudate. Petals 9-11, white, occasionally pink or light purplish red suffused at base, obovate, apex emarginate. Stamens numerous. Anthers yellow; filaments pale red-purple; disc pale red-purple. Carpels 5, densely coarse tomentose. Stigmas pale purplish red. Follicles 5, thickly brown-gray rough tomentose. Seeds black and glossy. Flowering middle or late April to early May. Fruits August (Fig. 1-23).

The species is found in mountainous thickets in alt. 1,200 m of Yangshan of Songxian County, Henan Province. P. ostii was first reported by Hong T. and Zhang J.X. in 1992. Except for Yangshan, it is also distributed in Lushi, Henan; Longshan, Hunan; Liuba, Shaanxi; Baokang, Hubei; Liangdang, Gansu; Chaohu and Ningguo, Anhui. This species have been cultivated as traditional Chinese medicinal plant for a long time. 'Feng Dan Bai' is the main cultivar used for a medicine and as well as an ornamental plant. It produced a series of cultivars of "Feng Dan" with higher resistance to heat and humidit. According to the author's observation, the cultivars derived from "Feng Dan" generally grow well in Pengzhou of Sichuan, Shanghai, Nanjing of Jiangsu, Hangzhou of Zhejiang etc. It is an important germplasm resource to breed new cultivars resistant to heat and wet.

P. rockii (S.G. Haw & L.A. Lauener) T. Hong et J.J. Li (P.papaveracea Andr., P. suffruticosa Andr. var. papaveracea (Andr.) Kerner, P. suffruticosa Andr. subsp. rockii S.G. Haw & L.A. Lauener)

Deciduous shrub up to 1.5 m tall. Stems straight, base flake scabbard. Proximal leaves 2- or 3-pinnate compound, long petiolus.

Leaflets (15-) 19-70, ovate-oblong or oblong-orbicular lanceolate, apex rapid acuminate, above glabrous or long white villous on main vein, below few villous, more on vein. Sepals 4, nearly orbicular, apex short acute caudate; petals ca. 10, commonly white, occasionally pale pink and red; blotches big blackish purple at the base above; stamens numerous; anthers yellow and filaments yellow-white; disc leathery scabbarded, white-yellow, enveloping ovary. Carpels 5, ovary densely short yellow wirehaired. Styles white-yellow; stigmas flat and plain. Follicles thickly short villous, apex beaked. Flowering May, fruits August.

This species has been differentiated into two subspecies with differences in morphology and geographical distribution.

P. rockii subsp. rockii (P. rockii subsp. linyanshanii T. Hong et G.L. Ostij

The leaves of this subspecies are ovate-elliptic or long-orbicular-lanceolate (Fig. 1-24). It is distributed in the mountainous deciduous forest or thickets at altitudes of 1,100 - 2,800 m in south Gansu, southern slopes of Qinling in Shaanxi, the mountains of Funiushan in Henan and Shennongjia in Hubei.

P. rockii subsp. taibaishanica D.Y. Hong

The leaflets of this subspecies are ovate or wide ovate and segmented or lobed. It is located in northern slopes of Qinling, Mt. Taibai,

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Figure 1-23 (left) P. ostii

Figure 1-24 (right) P. rockii

Longxian, Ganquan and Fuxian in Shaanxi; North Xiaolongshan, West Qinling, Middle Ziwuling (Heshui) in Gansu (Fig. 1-25,Fig. 1-26). It is the wild original species of Xibei (northwest) group of tree peony cultivars. This species is used as medicinal and ornamental plant. The wild population is dramatically decreasing and almost disappearing because of overdigging.

The author found pink and red flower types of P. rockii during investigating in the forests of Ziwuling in East Gansu and North Shaanxi. (See 9.1 of Chapter 9). These wild varieties were also found by the local forestry workers and druggists in Wudu and Kangxian, South Gansu. Unfortunately, they could not collect the specimens with flower because it was not the blossoming season during that time. Besides the variation of the flower color, the blotches of the petals, the shapes and color of leaves are variable, too. P. rockii with red blotches flowers was found in Shennongjia of Hubei, too (Fig. 1-27).

1.2.2 Reasons Causing Wild Tree Peonies Endangered and Their Protection

A Continued Decline Trend of Distribution Area in Wild Tree Peony

According to the ancient literatures, tree peonies were widely located in China. In these records, Xie L.Y. (AD385-433) in the Jin Dynasty (AD265-420), as the earliest describer of tree peony, recorded: "There are many tree peonies between bamboos along the riversides of Yongjia". Latter, in the book of Xin Xiu Ben Cao (AD657) in the Tang Dynasty (AD618-907), it recorded that tree peonies grew in Jiannan, Hanzhong and Bajun etc. in Sichuan. Su S. (AD1020-1101), in the Song Dynasty (AD960-1279), said in Tu Jing Ben Cao (AD 1061), "Tree peonies grow in the valleys of Bajun and Hanzhong, now they are found in the mountains of Danzhou, Yanzhou, Qingzhou, Yuezhou, Chuzhou and Hezhou. The flowers are yellow, purple, red, and white etc. These should be the wild tree peonies". Besides, Su S. mentioned that tree peonies were also distributed in Hezhou and Xuanzhou. In the Bei Song Dynasty (AD960-1127), Ouyang X. (AD1007-1072) mentioned in his book Luo Yang Mu Dan Ji: "Tree peonies are so common in the slopes of the west of Danzhou and Yanzhou that local people use them as the firewood". According to the above

records, the distributional areas of tree peonies should be:

—The west of Danzhou and Yanzhou now equal to the regions of Yichuan, Yan' an and the uplands of the Loess Plateau;

—Qingzhou equal to the regions of Zibo and Yidu in the middle of Shandong;

—Yuezhou equal to the middle and north of Zhejiang;

—Chuzhou, Zhuzhou and Xuanzhou equal to the east and SE Anhui;

—Hanzhong and Baoxiedao equal to the uplands of Qinling and Daba in the south of Shaanxi;

—Jiannan equal to the west of Sichuan, the NE of Yunnan and the south of Gansu;

—Bajun equal to east of Sichuan, Hezhou equal to Hezhou City and its surroundings of Chongqing. Most of the distributional areas of wild tree peonies mentioned above have been proved recently. Although only a few natural vegetations now remained in Shandong Province, the wild tree peonies were still possibly distributed there during the ancient times. The wild P. ostii was most probably located in SE Anhui and North Zhejiang. The original species used as medicine in Fenhuangshan, Tongling of Anhui is P. ostii. The specimens of this species were collected by Shen B.A. (1997) in Ningguo of Anhui. In addition, wild white-flowered tree peonies in Anhui were recorded in Chao Xian Zhi and Wu Wei Xian Zhi. Hong T. and Zhang J.X. thought the old wild tree peony with white flowers was P.ostii, too, which was found on the cliff of Yinpingshan in Chaoxian. Although wild tree peonies were widely located in China in ancient time, the current distributional areas of them have dramatically declined. For example, in the middle and south of Gansu, more than 2/3 of the distributional areas of P. rockii have disappeared and this species became extinct in Maxianshan (Li JJ. & Chen D.Z. , 2002). In the!950's, wild tree peonies could be found in the uplands of Songxian, Luanchuan, Yiyang, Luoning, Xin'an, Mianchi, Lingbao, Lushi, Ruyang, Linru etc. in the west of Henan province, but at present only a few can be found in Songxian, Luanchuan, Lushi and its south side. P. rockii is nearly extinct in Mt.Taibai of Qinling. Wild tree peonies are mostly distributed in island patterns and each population usually consists of only a few plants.

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Figure 1-25 P. rockii subsp. taibaishanica

Figure 1-26 P. rockii under deciduous broad-leaved

Figure 1-27 WildP. rockiiwith redblotches forest (Ziwuling, Gansu) of Shennongjia, Hubei

Major Reasons Causing the Wild Tree Peony Endangered

The habitat of the wild tree peonies is seriously threatened by the increasing human activities. In 1987, P. jishanensis, P.rockii and P.lutea were considered as the Chinese national third-class preserved plants in The List of National Rare and Endangered Plant Species. Since then, the investigations of tree peonies have been conducted throughout China. Based on the investigations and researches, there are many reasons to cause wild tree peonies endangered, including both internal and external factors, especially effect of human activities. On the other hand, the endangered extent is different among the wild species.

The Speicific Biology Characters In a natural population, tree peonies grow slow. It takes 7 years in average from seed to blossoming and bearing fruits. During sexual reproduction, many disadvantage factors will affect the propagation of tree peonies, especially the properties of the seed:

—Under natural conditions, the number of the flowered plants is small in a wild population. The same twig usually blooms only once every two years. For example, only 14% of the plants bloom in P. jishanensis and 30% - 50% in P. rockii. During blossoming, infertile pollen, ovule abortion (over 80% of ovule abortion rate in P. jishanensis) and the simple primitive surface structure of stigma will result in a low fruiting rate and less seeds.

—Most of wild tree peonies are distributed in island patterns. One population usually consists of few plants. The crossbreeding rate is low and the seed vitality is decreasing.

—The low rate of germination and rooting. For instance, the germination and rooting rate of P. rockii in Lueyang of Shaanxi and Shennongjia of Hubei is 4.4% and 12% respectively. Under natural conditions, the germination rate of P. jishanensis is only 4%.

—Tree peony seeds have characteristic of epicotyl dormancy. Suitable conditions are required to break dormancy. Besides the dormant character of embryo, the seeds of P. lutea are easily vulnerable to inactivation as water loses in dry weather.

—The damage of beetles. Beetles like to eat carpels and ovules during pollination, especially in P. rockii. In addition, seed pests take endosperm as food in P. lutea. The extent of damage is up to 90% in some places. For a certain species, the limitation of propagation usually resulted from an accumulative effect of many factors. For example, all these factors, such as pollen infertility, ovule abortion, the space restriction of carpel to ovule, low germination rate in natural populations and change of external environments, inhibit propagation and extension of P. jishanensis (Zhang S.Z. et al. 1997).

The Effects of Human Activities Human activities have imposed both detrimental and protective effects on wild tree peonies. The harmful effects mainly include two aspects: One is that wild tree peonies are not only rare ornamental flowers but also raw material of a Chinese medicine "Dan Pi". Overdigging has caused the extinction of many wild tree peonies resources. The other is that, with increasing human activities, large scales of natural vegetations were destroyed by such as disforestation and reclamation, excessive grazing and the indirect damages from making road, exploiting mine and so on, which

destroyed the habitat of wild tree peonies to different extents and deprived their growth and propagation spaces, and finally caused the wild tree peonies endangered.

Conservation of Wild Germplasm Resources of Tree Peonies At prestent, the protection of wild germplasm resources of tree peonies is under study. The three following effective measures should be:

—To widely publicize the importance of protecting wild resources and forbid digging wild tree peonies.

—To establish the protection area in core distribution area for in vitro conservation.

—To establish the national germplasm resource nursery to carry out ex-situ conservation, etc. The effect of the in-vitro preservation of wild tree peonies is obvious. For example, P. rockii is the most endangered species in the Taibai forest of Heshui in Ziwuling, Gansu. However, after 23 years of forbidding digging wild plants, the wild population of this species has been gradually recovering in the area of nearly 200 hm² which was badly destroyed before (Ding O.K. et al., 2002). For P. rockii, P. lutea and P. potaninii, they are distributed in NW Yunnan, SW Sichuan, SE Tibet and West Guizhou and mainly reproduce by subterraneous stems. The endangered situation can be avoided if only over-digging and illegal collecting are successfully prohibited.

1.3 Research and Use of Wild Germplasm Resources

1.3.1 The Introduction and Observation of the Wild Species

In order to widely collect, preserve and protect wild germplasm resources of tree peonies, after many years' hard work, the author has successfully established a germplasm resource nursery in Lanzhou

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involving the most species of Chinese wild tree peonies and performed cross-breeding and interdisciplined investigation here.

Lanzhou is located in the middle temperate (warmer) and semiarid climate zone. Irrigation is needed as a complement because of less rain here. Lanzhou is a high altitude region (the introduced areas in alt. 1,550 - 1,700 m). It is not hot in summer but cold in winter. The soil is a little alkaline (pH 8.0-8.5). Except P. ludlowii with less adaptability, all other introduced species can normally grow, bloom and fruit and have successfully experienced a life cycle from seed to seed. The "sprouting" phenomenon is usually found in P. ludlowii after winter, but some protective measures can easily make it pass winter. It grows vigorously but only forms few flower buds. Some introduced plants of P. rockii and P. lutea are obviously superior to the wild ones, however the annual growth rate of each species is nearly the same as that in the wild areas.

Except for P. jishanensis and P. ludlowii, the flowering rate of a single plant is obvious higher than that of the wild plants. The flower diameter tends to be larger and the fruit rate obviously increases. For example, in the original areas, we collected 74 seeds from 6 fruits of P. jishanensis. But for the introduced plants, we got 62 seeds from 2 fruits in 2002. The number of seeds is up to 8 - 10 per carpel in P. delavayi. Fruit pest is a big problem for the fruits of P. delavayi and P. lutea in the original areas, therefore the seeding rate is low, too. However, in the introduced area, no fruit pest appears. The seeds normally develop and the germination rate is obviously superior to that of the original areas (Table 1-1).

1.3.2 The Blossoming Biological Characters of Wild Tree Peony Species

Phenological Observation

On the whole, the phenological period of the introduced plants is earlier than that of the original ones, although some differences are seen in interspecific and intraspecific wild tree peonies.

Generally, the sprouting period is on March 10th - 20th, but P. potaninii is the latest in late March. The primary flowering period is from April 1st (P. delavayi) to May 4th (P. potaninii). The time of forming terminal bud after flowering and the deciduous period of the Subsection Vaginatae are earlier (the terminal bud formed earliest on July 5th and leaf fallen in middle October) than the Subsection Delavayanae (the terminal bud formed in middle July, P. ludlowii is the latest after September; leaf fallen beyond middle November.)

The Characters of Flowering

During blossoming, characters of species and intraspecific differences appear. The differentiation of styles and stigmas in the Subsection Delavayanae is not as good as that of the subsection Vaginatae (see Chapter 1,1.1). The stigmas of Subsect. Vaginatae secrete much more mucilage than Subsect. Delavayanae and it is nearly invisible for the latter. According to the maturity time of stamen and pistil, the stamen matures earlier in Subsect. Vaginatae except for P. ostii. Pistils and stamens simultaneously mature in Subsect. Delavayanae and P. ostii. Self-pollination tests of P. jishanensis, P.ostii and P. delavayi, indicated that all the offspring can bear fruits but the fruiting rate is low. Therefore, the sexual reproduction of these species is probably mainly through cross-pollination within the same plant and between different plants in the wild.

Based on a 4-year investigation of growth and development characters of root, stem, leaf, flower, fruit, seed etc. in the introduced species, it suggested that all kinds of characters of wild species are quite stable and the same as those in the original areas under the same conditions. From this point, the characters of wild species and different populations are apparently determined by genetic information (genotype). Ex-situ conservation successfully protects the genetic diversity of germplasm resources.

Table 1-1 The growth and development contrast of wild tree peonies between original areas and introduced areas

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Figure 1-28 The pollen exine patterns of genus Paeonia section Moutan

1-2 P. qiui (Baokangpop.)

3-6 P. jishanensis

3-4 P. jishanensis (Jishanpop.)

5-6 P. jishanensis (Yan'anpop.)

7-8 P. ostii (Yangshan pop.,Song County)

9-14 P. rockii

9-10 P. rockii (Yangshanpop.)

11-12 P. rockii (Taibaishan pop.)

13. P. rockii (Maxianshan pop.)

14. P. rockii (Wenxian County pop.)

15-16 P. decomposita (Jinchuan pop.)

Figure 1-29 The pollen exine patterns of genus Paeonia Section Moutan

1. P. decomposita (Ma 'erkang pop.)

2. P. jishanensis (Jishan pop.)

3. P. qiui (Baokang pop.)

4. P. ostii (Yangshan pop., Song County)

5-6. P. rockii (Baokangpop.)

7. P. ludlowii

8. P. potaninii (Yajiangpop.)

9. P. delavayi (Annan pop., Zhongdian)

10. P. lutea var. alba

11. P. lutea (Tangxiong pop., Zhongdian)

12. P. lutea x P. delavayi

13. P. delavayi (Yulongxushan pop.)

14-15. P. lutea x P. delavayi (No.14 from orange flower, No.15 from red)

1.3.3 The Analysis of Genetic Diversity

The Study in Palynology

The characters of pollen granule exine are regarded as one of the most valuable classification evidence in palynological studies, the variation extent of the exine of pollen granule usually determines the evolutional level of a taxon. The pollen granule exine of genus Paeonia Section Moutan is complex and variable (Xi Y.Z. , 1984; Yuan T. , 2000; He L.X. et al. in press). The author found that the exine of pollen granule expressed complex features at the population level in this subsection (Fig.1-28,1-29): (1) In Subsect. Vaginatae, the foveolate or reticular exine in Jishan pop. of P. jishanensis and foveolate or foveolate-reticulate in Yan'an pop.; Small foveolate or irregular stripe-reticular exine in Baokang pop. of P. qiui and foveolate-reticulate for Shennongjia pop.; Rugate undulate-reticular exine in Baokang pop.of P. ostii arid foveolate-reticulate or reticulate for Yangshan pop.; The reticular exine in Ziwuling pop. of P. rockii, rugate undulate-rough reticular for Lintao pop., reticular or rough reticular for Wenxian pop. and rough reticular for the populations of Yangshan, Taibaishan, Maxianshan, Dangchuan and Baokang, the biggest mesh of exine in Baokang pop.; The thin reticular or foveolate exine in P.decomposita. (2) In the Subsection Delavayanae, the foveolate-reticulate exine in P. ludlowii and P. potaninii, but biggest perforations shown in the latter; Rugate undulates exine in Yulongxueshan pop. of P. delavayi and reticular for Annan pop.; The rugate undulate-rough reticular exine in Ludian and Gezan populations of P. lutea, reticular in populations of Tangdui, Deqin and Zhongxianggonglu, and rugate undulate- reticular in the other populations of P. lutea.

According to the results above, it indicates:

—In Section Moutan, various patterns of the pollen granule exine appear, such as small foveolate, foveolate, foveolate-reticulate, reticular, rough-reticular, rugate undulates-reticular, rugate undulatus patterns etc.. The shapes and sizes of pollen grains are also different in this section.

—The pollen exine veins may be the same or similar between the species with different external characteristics, such as Lintao pop. of P. rockii and Baokang pop. of P. qiui, Ziwuling pop. of P. rockii and Shennongjia pop. of P. qiui, Annan pop. of P. potaninii and Annan pop. of P. delavayi.

—The pollen exine veins of different populations are obviously different in a species because of heterogeneous environmental conditions, which resulted in the polymorphism of the exines in the same species. From the evolutional principles of pollen exine, including isochronous evolution of rugate undulate and reticular veins, we may speculate that a

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Figure 1-30 The Chromosomes of Genus Paeonia Section Moutan

1. P. decomposita

2. P. jishanensis

3. P. qiui

4. P. ostii

5. P. rockii (Zhouqu pop.)

6. P. ludlowii

7. P. potaninii

8. P. delavayi (Lijiang pop.)

9. P. lutea

rough order of evolution is: P. ludlowii —> P. potaninii —> P. delavayi —> P. lutea in Subsection Delavayanae', P. decomposita —> P. qiui —» P. jishanensis —> P. ostii -> P. rocfo';' in Subsection Vaginatae.

The Karyotype Analysis of Chromosomes

In genus Paeonia Section Moutan, the karyotype has been considered quite stable and no differentiation for many years (Hong D.Y. et al 1988; Li J.J. et al. 1999). However, He L.X. et al. recently found obvious differentiations in different intraspe-cific populations (Fig. 1-30).

Different Karyotypes between Annan and Lijiang Populations in P. delavayi.

The karyotype formulation of Annan pop. is 2n=2x =10=4m+4sm+2st, namely, there are 4 sub-metacentric chromosomes and the karyotype is more dissymmetrical. Chromosome fragments were found in 31.25% of cells and the supernumerary chromosome (B chromosome) may appear in this population. It may be one of the reasons of strong adaptability, high viability and easy flower formation for Annan pop. of P. delavayi.

The Kayotype Variation of P. rockii.

The karyotype formulation of Baokang pop. is 2n=2x=10=6m+3sm+lst, with one sub-metacentric chromosome and absence of one sub-acrocentric chromosome.

The karyotype formulation of Lintao pop. is 2n=2x=10=6m+2sm(2SAT)+2st (2SAT). There are two special sub-metacentric chromosomes, one is the longest and the other is the shortest. The length ratio of these two chromosomes is 2.34. The chromosomes with arm ratio more than 2:1 are 20%. According to the Stebbins' classification standard of symmetry karyotype, it belongs to 2B karyotype, which possibly resulted from the Robertsonian Translocation.

The karyotype formulation of Zhouqu pop. is 2n=2x=10=4m+6st. The chromosome ratio of the longest to the shortest is greater than 2. The arm ratios of over 50% of the

chromosomes are greater than 2:1. It should belong to 3B karyotype.

The karyotype formulation of Baokang pop. of P. qiui is 2n=2x=10=8m+2st. It is more primitive than that of Shennongjia pop. with the karyotype formulation of 2n=2x=10=6m+2sm+2st, which was reported by Y.L. Pei in Shennongjia pop. (Obvious differences between the two populations were also found in palynology analysis).

The morphological characters of wild populations of P. lutea greatly vary as well as the chromosome karyotypes. X. Gong et al. (1997) mentioned 4 karyotype patterns through the investigation of 7 populations of P. lutea.

—The karyotype formulation of Liangwangshan and Tuguancun populations is 2n=2x= 10=6m+2sm+2st.

—The karyotype formulation of Xishan pop. is 2n=2x=10=6m+2sm(S AT)+2st(S AT).

—The karyotype formulation of Ludian pop. is 2n=2x=10=5m+2sm+3st(SAT).

—The karyotype formulation of Zhuoganshan, Nixi, Wengshui populations is 2n=2x=10=7m+lsm+2st(2SAT). Through the observation of 4 populations, the author also obtained the similar results: the karyotype formulation of Tangdui pop. of Zhongdian is similar to (4); the karyotype formulation of Halacun pop. of Zhongdian is the same as (1); the karyotype formulation of red-flowered Halacun pop. of Zhongdian is 2n=2x=10=6m+2srn (2SAT)+2st(2SAT). Other researchers (Li S.F. et al., Yang D.Q. et al. 1989) reported different karyotypes in different populations, too. Besides, Xiao T.J. et al. (1997) and Gong X. et al. found that the patterns of Giemsa C-band were more variable based on the investigation of 10 populations of P. lutea. It indicated that the genetic differentiation have been occurred in these populations.

The Analysis of Protein Bands of Leaves and Seeds

From the analyses of the protein zone electrophoretograms of

leaves of 6 species and seeds of 7 species, it suggested that the specific characteristics of species are associated with the leaf protein bands (Yu L. et al., 1997). He L.X. et al. (in press) performed cluster analysis of seed protein bands using the shortest distance method. As a result, the subsection Delavayanae and Vaginatae converge into two big branches respectively. In Subsect. Delavayanae, two populations of P. lutea meet first and then converge with P. delavayi and P. potaninii in turn. In Subsect. Vaginatae, 3 populations of P. rockii meet first and then converge with P. ostii, P. jishanensis and P. decomposita in turn. According to the coefficient difference of protein zones, it is obvious higher in the pecies of Subsect. Dalavayanae than in those of Subsect. Vaginatae. Especially P. potaninii, with the most seed protein bands of 39, is much more distinguished from other species.

The coefficient difference between P. potaninii and P. rockii of Baokang is the highest up to 54.8. It is also a little higher namely 31.3-40.0 between P. potaninii and P. delavayi, P. lutea. Comparing with the seedlings between the original areas and the introduced area, however, the coefficient of difference of seed protein zones

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of P. lutea is only 11.5. As for the characters of seedling descendants, the difference is not found in the introduced area within Subsect. Delavayanae. It indicates that the heterozygosis dose not appear in this section and this phenomenon may provide an important clue to systematic classification.

Other Informations

Zou Y.P. et al. (1999b) used RAPD molecular marker to study the genetic diversity of 4 populations (Jishan, Yongji, Tongchuan and Huashan) in P. jishanensis and analyzed the data by AMOVA (analysis of molecular variance). He found that the polymorphism loci of the 4 populations occupy 67.7% of all amplified fragments. It indicates that genetic diversity tends to be high as a whole. The inheritable variation of inter-populations (52%) is greater than that of intra-populations (48%). A big differentiation occurs between Tongchuan population and others and the interpopulation genetic distance positively correlates with geography distance.

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