Journal of Plant Research
© The Botanical Society of Japan and Springer-Verlag  2003
10.1007/s10265-003-0130-6

Original Article

Chemical taxonomy of the Xibei tree peony from China by floral pigmentation

Liang-Sheng Wang1, Fumio Hashimoto1 Contact Information, Aya Shiraishi1, Noriaki Aoki2, Jia-Jue Li3 and Yusuke Sakata1

(1)  Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan
(2)  Faculty of Life and Environmental Science, Shimane University, Matsue, Japan
(3)  Faculty of Environmental and Chemical Engineering, Luoyang University, Luoyang, China

Contact Information Fumio Hashimoto
Email: fhashimo@farm.agri.kagoshima-u.ac.jp
Phone: +81-99-2858559
Fax: +81-99-2858559

Received: 3 June 2003  Accepted: 20 October 2003  Published online: 18 December 2003

Abstract  Petal flavonoid compositions of 39 tree peony cultivars from Xibei (northwest China) were investigated in order to study the chemotaxonomic relationship among tree peony species. Six anthocyanins, the 3-O-glucosides and 3,5-di-O-glucosides of three anthocyanidins—pelargonidin (Pg), cyanidin (Cy), and peonidin (Pn)—exist in petals without a blotch at the base. The flowers are classified into three anthocyanidin phenotypes: lsquoPn, Pg>Cyrsquo; lsquoPn, Cyrsquo; and lsquoPn, Cy>Pgrsquo. Furthermore, the yellow pigments are identified as three flavones and three flavonols: apigenin, luteolin, chrysoeriol, and kaempferol, quercetin, and isorhamnetin, respectively. Wardrsquos minimum-variance cluster analysis with principal component analysis produced a dendrogram using standardized scores of 20 pigment variables. Of 39 cultivars, 11 clustered with white flowered Paeonia rockii and 17 with pink flowered P. rockii. The other 11 cultivars matched either P. delavayi or P. potaninii. The result suggests that the Xibei tree peony originated mainly from P. rockii.

Keywords  Anthocyanin - Flavonoid - Flower color -  Paeonia  - Principal component analysis - Tree peony

Introduction
In China, the tree peony (Paeonia spp., Chinese mudan) has been named the lsquoking of flowersrsquo, bringing good fortune and happiness. Chinese tree peony cultivars are geographically classified into four groups: (1) Zhongyuan cultivars, which are dwarf and have circular or elliptic leaves (in the Central Plains region along the middle to downstream sections of the Huanghe River, including cultivars from the cities of Luoyang, Heze and Beijing); (2) Xibei cultivars with a distinctive blotch at the base of the petals (Gansu, Qinghai, Shaanxi and Ningxia Provinces in Northwestern China); (3) Jiangnan cultivars, mainly consisting of lsquoFeng Danrsquo Moutan, of which the root bark is used as Chinese medicine; and (4) Xinan cultivars from Sichuan and Yunnan Provinces (Li 1989; Qin 1997) (Fig. 1). In the Tang dynasty (AD 618–907), Xibei tree peony became a famous ornamental plant and was widely distributed in most areas of Gansu Province. There are now over 100 cultivars reported in this group (Li 1989; Chen et al. 1994).
MediaObjects/s10265-003-0130-6fmb1.gif
Fig. 1  The distribution of four groups of Chinese tree peony cultivars based on their morphological characteristics and producing areas

The Xibei tree peony group has a conspicuous, distinctive morphological characteristic, a clear black-purple or purple-red blotch at the base of the petals. Since over 30% of Zhongyuan tree peony cultivars also have a blotch of the same color at the base of the petals, it was believed that cultivars might often have been exchanged between the two regions (Li 1989).

It has been reported that there exist six anthocyanins in tree peony flowers as follows:
–  peonidin 3,5-di-O-glucoside (Pn3G5G);
–  pelargonidin 3,5-di-O-glucoside (Pg3G5G) (Hayashi 1939, 1943);
–  cyanidin 3,5-di-O-glucoside (Cy3G5G) (Hayashi and Abe 1953);
–  pelargonidin 3-O-glucoside (Pg3G) (Cooper 1970);
–  peonidin 3-O-glucoside (Pn3G) (Ishikura and Sugawara 1979); and
–  cyanidin 3-O-glucoside (Cy3G) (Kato 1982).

Hosoki et al. (1991) investigated 21 Chinese tree peony cultivars to identify six anthocyanins by thin layer chromatography. Sakata et al. (1995, 1996) established simultaneous method for determination of six anthocyanins in tree peony flowers by high performance liquid chromatography (HPLC) analysis.

Our laboratory has first identified the presence of six flavone and flavonol aglycones (Wang et al. 2001b). Apigenin, luteolin, kaempferol, and quercetin were identified by comparison with the data obtained from authentic samples. Furthermore, methylated flavonoids, chrysoeriol and isorhamnetin, were identified for the first time by nuclear magnetic resonance spectra. Recently, we have represented the phenetics in tree peony species from China by flower pigment cluster analysis (Wang et al. 2001b). The objective of the present study was to focus on the chemotaxonomic relationship with the original tree peony species by using floral pigment clustering in order to provide data for phylogenic positioning of tree peony cultivars.

Materials and methods
Plant materials
Petals of 39 cultivars of Xibei tree peony (Table 1) were collected from the end of April to the middle of May in 1998 and 1999. Most of the petals were obtained from the Yuzhong Peace Peony Garden, Yuzhong prefecture; otherwise, petals were collected from the Garden of Ning-Wo-Zhuang Hotel, Lanzhou City and the parterre of Northwest Normal University, Lanzhou City, Gansu Province, China.
Table 1  Petal coloration of 39 Xibei tree peony cultivars

Cultivara

RHSCCb

CIELab coordinatec

h d

L*

a*

b*

C*

lsquoPn, Pg>Cyrsquo (3 cultivars)

  Pink flowered

    Fen Jin Yu

62D

84.0

12.0

3.0

12.4

14.0

    Fen Yan Jiao

62D

82.2

14.1

–1.7

14.2

–6.9

  Purple flowered

    Lan Xian Nu

75D

82.3

14.9

0.4

14.9

1.5

lsquoPn, Cyrsquo (31 cultivars)

  Pink flowered

    Jin Cheng Wan Xia

62D

84.0

8.9

6.7

11.1

37.0

    Jin Cheng Nu Lang

63C

65.1

35.4

1.3

35.4

2.1

    Hui Die

65D

70.1

31.0

–2.3

31.1

–4.2

  Purple flowered

    Mei Gui Sa Jin

71A

29.1

65.6

–5.3

65.8

–4.6

    Zi Guan Yu Dai

71A

27.7

62.9

–8.8

63.5

–8.0

    Li Chun

72B

35.4

67.6

–15.6

69.4

–13.0

    Yan Yun Liao Rao

72B

37.4

60.7

–11.6

61.8

–10.8

    Bing Xin Hong Lian

72B

41.8

58.2

–19.2

61.3

–18.3

    Jin Cheng Hong

72B

48.3

59.2

–14.7

61.0

–13.9

    Mo Ai

72B

47.2

57.9

–16.6

60.2

–16.0

    Jing Shen Huan Fa

74B

51.9

53.1

–14.6

55.1

–15.4

    Fan Tao Hui

75A

59.0

44.2

–13.2

46.1

–16.6

    Hong Lian

75B

65.3

35.8

–13.1

38.1

–20.1

    Jin Yu Xi Shui

75B

63.7

36.9

–12.1

38.8

–18.2

    Qing Shen

77B

46.1

57.5

–21.6

61.4

–20.6

    Hong Guan Yu Zhu

77C

55.2

50.3

–18.9

53.7

–20.6

    Lan Feng Zhan Zhi

77D

60.1

42.1

–16.7

45.3

–21.6

    Lan He

77D

69.3

33.9

–10.4

35.5

–17.1

    Zhong Shan Feng Yu

78B

50.0

54.4

–18.1

57.3

–18.4

    Hong Zhuang Su Guo

78B

50.1

52.8

–19.9

56.4

–20.7

    Hong Hai Yin Bo

80B

50.2

54.3

–16.7

56.8

–17.1

  White flowered

    Xue Hai Bing Xin

W

90.3

–1.5

12.4

12.5

96.9

    Qi Lian Cai Hong

W

88.7

–0.7

13.7

13.7

92.9

    Yi Dian Mo

W

88.4

–0.9

9.4

9.4

95.5

    Fen Yu

W

88.5

2.6

6.6

7.1

68.5

    Bai He Liang Zhi

W

88.5

2.3

6.1

6.5

69.3

    Tao Hua Fen

W

88.3

0.4

9.0

9.0

87.5

    Zhuan Bian Huang

4D

90.0

–4.7

18.7

19.3

104.1

    Huang Lian

4D

90.6

–1.9

11.9

12.1

99.1

  Black flowered

    Ye Guang Bei

59A

23.5

72.8

2.8

72.9

2.2

    Hei Tian E

59A

24.5

67.3

4.8

67.5

4.1

lsquoPn, Cy>Pgrsquo (3 cultivars)

  Purple flowered

    Chun Hong Zheng Yan

75D

80.1

14.6

–0.9

14.6

–3.5

  White flowered

    Yu Ban Xiu Qiu

W

88.8

–1.2

11.0

11.1

96.2

    Yu Rong Dan Xin

4D

90.5

–3.0

14.7

15.0

101.5

Others (2 cultivars)

  White flowered

    Shu Sheng Peng Mo

W

89.4

0.3

9.0

9.0

88.1

    Xiao Xue

W

90.5

0.2

5.9

5.9

88.1
aCultivars were classified by anthocyanidin contents as follows: Pn peonidin, Cy cyanidin, Pg pelargonidin (Wang et al. 2001a)
bRoyal Horticultural Society Colour Chart; W white color
c L * Lightness; a * , b * chromatic components; C * chroma (brightness)
d h Hue angle=arctan(b*/a*) (degrees)
Nine species and one variety are identified as wild tree peony (Li and Li 1997; Hong and Pan 1999). We collected petals of seven wild species with one variety in Yunnan, Sichuan and Gansu Provinces, and recently reported petal pigment constitution and their phylogenic relationship (Wang et al. 2001a). The wild species used in this study are divided into two subsections, Vaginatae and Delavayanae. The former included Paeonia jishanensis, P. ostii, P. ostii var. lishizhenii, P. qiui, P. rockii and P. decomposita, and the latter included P. potaninii and P. delavayi. The locations of collection are summarized in Fig. 2. The data for eight accessions are included in Tables 2 and 3.
MediaObjects/s10265-003-0130-6flb2.gif
Fig. 2  Locations of the eight accessions of wild tree peony collected in China. 1 Paeonia jishanensis (white), Government-owned forest, Jishan, Shanxi; 2 P. ostii (white), Mt. Yangshan, Song Prefecture, Henan; 3 P. qiui (pink), Mt. Laoyashan, Baokang, Hubei; 4 P. rockii (pink), Shaba, Mt. Xiaolongshan, Gansu; 5 P. rockii (white), Shangdanbao, Wen Prefecture, Gansu; 6 P. decomposita (pink), Jimu, Jinchuan, Sichuan; 7 P. potaninii (purplish red, reddish purple), Bajiaolou, Yajiang, Sichuan; 8 P. delavayi (blackish purple), Xianjingya, Lijiang, Yunnan
Table 2  Petal anthocyanin composition of Xibei tree peony cultivars (39 cultivars ) and tree peony species

Cultivar or species

Pna

Cya

Pga

Aglyconea

Glycosidea

TAb

3G5G

3G

3G5G

3G

3G5G

3G

Pn

Cy

Pg

3G5G

3G

lsquoPn, Pg>Cyrsquo (3 cultivars)

  Pink flowered

    Fen Jin Yu

64

5

+

13

18

–

69

13

18

82

18

0.032

    Fen Yan Jiao

76

+

4

+

20

–

76

4

20

100

+

0.035

  Purple flowered

    Lan Xian Nu

41

+

+

29

30

–

41

29

30

71

29

0.007

lsquoPn, Cyrsquo (31 cultivars)

  Pink flowered

    Jin Cheng Wan Xia

86

+

14

+

–

–

86

14

–

100

+

0.016

    Jin Cheng Nu Lang

87

2

9

2

–

–

89

11

–

96

4

0.30

    Hui Die

64

9

5

22

–

–

73

27

–

69

31

0.08

  Purple flowered

    Mei Gui Sa Jin

55

19

16

10

–

–

74

26

–

71

29

3.44

    Zi Guan Yu Dai

37

11

24

28

–

–

48

52

–

61

39

2.19

    Li Chun

73

3

22

2

–

–

76

24

–

95

5

1.76

    Yan Yun Liao Rao

68

9

20

3

–

–

77

23

–

88

12

1.20

    Bing Xin Hong Lian

83

3

14

+

–

–

86

14

–

97

3

0.79

    Jin Cheng Hong

79

3

11

7

–

–

82

18

–

90

10

0.75

    Mo Ai

77

3

16

4

–

–

80

20

–

93

7

1.25

    Jing Shen Huan Fa

78

5

14

3

–

–

83

17

–

92

8

0.84

    Fan Tao Hui

80

2

9

9

–

–

82

18

–

89

11

0.46

    Hong Lian

65

6

17

12

–

–

71

29

–

82

18

1.22

    Jin Yu Xi Shui

88

+

9

3

–

–

88

12

–

97

3

0.12

    Qing Shen

78

2

15

5

–

–

80

20

–

93

7

1.19

    Hong Guan Yu Zhu

94

–

6

–

–

–

94

6

–

100

–

0.36

    Lan Feng Zhan Zhi

84

–

16

–

–

–

84

16

–

100

–

0.18

    Lan He

86

–

9

5

–

–

86

14

–

95

5

0.058

    Zhong Shan Feng Yu

87

+

13

+

–

–

87

13

–

100

+

0.30

    Hong Zhuang Su Guo

85

–

13

2

–

–

85

15

–

98

2

0.56

    Hong Hai Yin Bo

69

7

16

8

–

–

76

24

–

85

15

0.90

  White flowered

    Xue Hai Bing Xin

70

6

18

6

–

–

76

24

–

88

12

0.010

    Qi Lian Cai Hong

87

+

13

+

–

–

87

13

–

100

+

0.011

    Yi Dian Mo

–

3

–

97

–

–

3

97

–

–

100

0.002

    Fen Yu

67

+

+

33

–

–

67

33

–

67

33

0.003

    Bai He Liang Zhi

83

5

5

7

–

–

88

12

–

88

12

0.004

    Tao Hua Fen

63

26

11

+

–

–

89

11

–

74

26

0.011

    Zhuan Bian Huang

55

–

+

45

–

–

55

45

–

55

45

0.004

    Huang Lian

42

12

18

28

–

–

54

46

–

60

40

0.017

  Black flowered

    Ye Guang Bei

39

17

16

28

–

–

56

44

–

55

45

4.19

    Hei Tian E

70

6

17

7

–

–

76

24

–

87

13

2.13

lsquoPn, Cy>Pgrsquo (3 cultivars)

  Purple flowered

    Chun Hong Zheng Yan

70

5

3

20

2

–

75

23

2

75

25

0.075

  White flowered

    Yu Ban Xiu Qiu

77

7

+

84

2

–

14

84

2

9

91

0.003

    Yu Rong Dan Xin

7

3

13

4

–

3

80

17

3

90

10

0.003

Others (2 cultivars)

  White flowered

    Shu Sheng Peng Mo

–

–

–

100

–

–

–

100

–

–

100

0.008

    Xiao Xue

–

–

–

–

–

–

–

–

–

–

–

–

Subsect. Vaginatae

    Paeonia jishanensis

82

–

5

–

13

–

82

5

13

100

–

0.16

    P. ostii

100

–

–

–

–

–

100

–

–

100

–

0.03

    P. qiui

90

–

10

–

–

–

90

10

–

100

–

0.26

    P. rockii (pink)

87

5

+

8

–

–

92

8

–

87

13

0.15

    P. rockii (white)

58

+

+

42

–

–

58

42

–

58

42

0.06

    P. decomposita

97

–

2

–

1

–

97

2

1

100

–

0.54

Subsect. Delavayanae

    P. potaninii

20

9

21

50

–

–

29

71

–

41

59

10.52

    P. delavayi

59

11

15

15

–

–

70

30

–

74

26

5.54
a Pn Peonidin; Cy cyanidin; Pg pelargonidin; 3G5G 3,5-di-O-glucoside; 3G 3-O-glucoside. Data are expressed as percentages
b TA Total anthocyanins (mg/100 mg dry petal)
Table 3  Petal composition of yellow pigments in Xibei tree peony cultivars (39 cultivars) and tree peony species

Cultivar or species

Flavonol and flavone aglyconesa

TFb

CIb

Km

Qu

Is

Ap

Lu

Ch

lsquoPn, Pg>Cyrsquo (3 cultivars)

  Pink flowered

    Fen Jin Yu

77

–

3

20

–

–

5.50

172

    Fen Yan Jiao

75

–

2

23

–

–

5.35

153

  Purple flowered

    Lan Xian Nu

82

–

2

16

–

–

7.88

1,126

lsquoPn, Cyrsquo (31 cultivars)

  Pink flowered

    Jin Cheng Wan Xia

9

24

39

7

14

7

4.83

302

    Jin Cheng Nu Lang

43

7

22

17

7

4

3.10

10

    Hui Die

13

19

24

14

23

7

7.82

98

  Purple flowered

    Mei Gui Sa Jin

25

25

17

14

17

2

11.5

3

    Zi Guan Yu Dai

16

18

14

11

36

5

6.01

3

    Li Chun

23

20

14

17

23

3

6.33

4

    Yan Yun Liao Rao

19

15

17

17

29

3

7.27

6

    Bing Xin Hong Lian

25

20

29

12

10

4

6.76

9

    Jin Cheng Hong

15

10

16

18

33

8

6.64

9

    Mo Ai

14

7

16

15

40

8

5.58

4

    Jing Shen Huan Fa

41

14

23

13

7

2

6.29

7

    Fan Tao Hui

16

9

20

18

26

11

6.85

15

    Hong Lian

15

26

18

9

28

4

5.92

5

    Jin Yu Xi Shui

19

10

16

15

31

9

5.75

50

    Qing Shen

24

14

26

11

16

9

8.65

7

    Hong Guan Yu Zhu

34

5

15

21

19

6

5.64

16

    Lan Feng Zhan Zhi

18

21

18

17

19

7

6.97

39

    Lan He

20

9

15

17

31

8

4.25

73

    Zhong Shan Feng Yu

11

20

27

14

23

5

6.71

23

    Hong Zhuang Su Guo

25

11

22

16

20

6

4.86

9

    Hong Hai Yin Bo

20

22

29

10

15

4

8.70

10

  White flowered

    Xue Hai Bing Xin

10

5

15

27

33

10

4.24

424

    Qi Lian Cai Hong

17

13

16

19

28

7

5.97

543

    Yi Dian Mo

64

–

2

34

–

–

5.78

2,890

    Fen Yu

75

–

3

22

–

–

6.36

2,120

    Bai He Liang Zhi

82

–

2

16

–

–

5.59

1,398

    Tao Hua Fen

82

–

2

16

–

–

4.82

438

    Zhuan Bian Huang

20

31

23

6

15

5

8.67

2,168

    Huang Lian

13

11

20

12

34

10

7.14

420

  Black flowered

    Ye Guang Bei

13

25

16

9

32

5

6.70

2

    Hei Tian E

14

27

18

7

31

3

5.96

3

lsquoPn, Cy>Pgrsquo (3 cultivars)

  Purple flowered

    Chun Hong Zheng Yan

64

3

7

22

3

1

5.05

67

  White flowered

    Yu Ban Xiu Qiu

64

–

3

33

–

–

4.85

1,617

    Yu Rong Dan Xin

17

24

29

8

16

6

6.47

2,157

Others (2 cultivars)

  White flowered

    Shu Sheng Peng Mo

69

–

3

28

–

–

4.82

603

    Xiao Xue

63

–

4

33

–

–

3.39

565

Subsect. Vaginatae

    Paeonia jishanensis

41

–

1

58

–

–

6.25

39

   P. ostii

36

+

2

57

3

2

3.12

104

   P. qiui

9

1

3

76

8

3

1.69

6

   P. rockii (pink)

13

15

34

15

15

8

8.31

55

   P. rockii (white)

21

4

3

70

–

2

4.83

80

   P. decomposita

8

1

4

68

5

14

3.28

6

Subsect. Delavayanae

   P. potaninii

8

13

11

24

41

3

4.38

0.4

   P. delavayi

10

51

37

–

2

–

10.75

1.9
a Km Kaempferol, Qu quercetin, Is isorhamnetin, Ap apigenin, Lu luteolin, Ch chrysoeriol. Data are expressed as percentages
b TF Total flavonols (mg/100 mg dry petal); CI copigment index=TF/TA

Pre-treatment of the fresh petals was as described in a recent paper (Wang et al. 2001b). Fresh petals were treated with boiling water for 2 s to prevent enzymatic degradation of pigments. The petals were allowed to dry overnight and kept in a desiccator for a week at room temperature to complete the drying process.

Flower color estimation

The measurement of flower coloration was performed as described in a recent report (Wang et al. 2001a). The color of fresh petals in the middle portion was measured according to the Royal Horticultural Society Colour Chart (RHSCC). The petal color of an individual was also measured at three locations with a NR-3000 color analyzer (Nippon Denshoku, Japan). This measurement is based on the Commission Internationale de lrsquoÉclairage (CIE D65/10°) scale (McGuire 1992; Voss 1992). CIE color data consist of a luminance or lightness component (L*), corresponding to the vertical axis, and two chromatic components: a* (from green to red) and b* (from blue to yellow). At L* values from 0 to 100, the blackness gradually decreases; the shade of green gradually decreases and the shade of red becomes more prominent from –a* to +a*. Moreover, from –b* to +b*, the tint of blue gradually diminishes and the yellow shade increases (CIE 1986). Chroma, C*, and hue (hue angle), h, were calculated according to the following equations: C*=(a*2 + b*2)1/2 and h=tan–1 (b*/a*) (Gonnet 1998). C* is the perpendicular distance from the lightness axis (more distance being more chroma). The hue angle is expressed in degrees.

Pigment extraction and HPLC analyses

The procedures of yellow pigment extraction and its acid hydrolysis were described in a recent report (Wang et al. 2001b). The dried petals (ca. 30–80 mg) were extracted with 1% HCl-MeOH (3 ml), then 2 N HCl (2 ml) was added to the extract, and the mixture was heated to 100°C for approximately 130 min in a heating block. The cooled reaction mixture was adjusted to 5 ml by adding 1% HCl-MeOH, directly filtered with Millipore disks (0.45 mgrm), and then submitted for HPLC analysis. Qualitative and quantitative HPLC analysis of three flavones and three flavonols was described in a recent report (Wang et al. 2001b). Qualitative HPLC analysis of flavone and flavonol aglycones was performed with a linear flow gradient under the following conditions: solution A consisted of H3PO4 and H2O in the ratio 1.5:98.5 (v/v); solution B consisted of H3PO4, HCO2H, CH3CN and H2O in the proportions 1.5:20:25:53.5 (v/v); the initial ratio of A to B was 65:35 (v/v), finishing at 20:80 (v/v) after 70 min; the column was TSKgel ODS-80Ts QA, internal diameter 4.5 mm, length 150 mm (Tosoh) at 40°C, with a flow rate of 0.8 ml/min, monitoring at a wave length of 370 nm and with an injection volume of 10 mgrl/sample.

The extraction of anthocyanins was done with acidic methanolic solution (CH3OH, H2O, HCO2H and CF3COOH in the proportions 70:27:2:1 [v/v]) (Hashimoto et al. 2000). Qualitative HPLC analysis of six anthocyanins was used with a linear flow gradient under the conditions described in a recent paper (Wang et al. 2001a). The HPLC for qualitative analysis was conducted with a linear flow gradient under conditions as follows: solution A consisted of H3PO4 and H2O in the ratio 1.5:98.5 (v/v); solution B consisted of H3PO4, HCO2H, CH3CN and H2O in the proportions 1.5:20:25:53.5 (v/v); the initial ratio of A to B was 80:20 (v/v), finishing at 40:60 after 60 min; the column was TSKgel ODS-80Ts QA, internal diameter 4.5 mm, length 150 mm (Tosoh) at 40°C, with a flow rate of 0.8 ml/min, monitoring at a wave length of 525 nm and with an injection volume of 10 mgrl/sample. The HPLC system was equipped with a multi-pump CCPM, auto-sampler AS-8000, column chamber CO-8011, detector UV-8010, power unit PS-8010 and super system controller SC-8010 (Tosoh).

Statistical analysis

The techniques of principal component analysis (PCA) and cluster analysis were used to classify the Xibei tree peony cultivars (Wang et al. 2001b). The calculation was done using the SAS system (SAS/STAT software, version 6.12, first edition; SAS Institute, Cary, N.C., USA) provided by the Kagoshima University Information Processing Center. The original data were chosen from the twenty variables Pn3G5G, Pn3G, Cy3G5G, Cy3G, Pg3G5G, Pg3G, Pn (peonidin), Cy (cyanidin), Pg (pelargonidin), 3G5G (3,5-di-O-glucoside), 3G (3-O-glucoside), Km (kaempferol), Qu (quercetin), Is (isorhamnetin), Ap (apigenin), Lu (luteolin), Ch (chrysoeriol), TA (total anthocyanins), TF ( total flavonols), and CI (copigment index). The eigenvalue and the eigenvector matrices were derived from PCA. According to the standardized values, the Euclidean distances among 39 cultivars were calculated. Based on the Euclidean distance, Wardrsquos minimum variance cluster analysis with semipartial R2 values was performed. The dendrogram of 39 Xibei cultivars with seven authentic species (eight accessions) was drawn using PROC TREE of the SAS system.

Results
Petal coloration

The RHSCC (Royal Horticultural Society Colour Chart) values of plain petals ranged from 59A to 80B including W and 4D (Table 1). Thirty nine cultivars of Xibei tree peony were classified into five groups according to the RHSCC: 15 cultivars belong to the red-purple group (59A–74B), 11 cultivars belong to the purple group (75A–78B), one cultivar was purple-violet (80B), 3 were light yellow (4D) and 9 white (W). There was no cultivar belonging to the red group.

The flower color was distributed on a CIELab color coordinate; the a* value ranged from –4.7 to 72.8, the b* value from –21.6 to 18.7, the L* value from 23.5 to 90.6, and the C* value from 5.9 to 72.9 (Table 1, Fig. 3). A significant correlation has been observed between L* and a* (L*=–0.008a*2–0.328a*+88.9, r 2=0.984).
MediaObjects/s10265-003-0130-6flb3.gif
Fig. 3  Distribution of Xibei tree peony cultivars by flower color based on CIELab data
Floral anthocyanins

Six anthocyanins—Pg3G, Pg3G5G, Cy3G, Cy3G5G, Pn3G and Pn3G5G— were detected by HPLC analysis from the plain part of petals. Two anthocyanidins, Pn and Cy, were found to exist in 37 cultivar flowers and most of them showed the lsquoPn>Cyrsquo phenotype except for three cultivars, namely, lsquoZi Guan Yu Dairsquo, lsquoYi Dian Morsquo and lsquoYu Ban Xiu Qiursquo (Table 2). Two cultivars, lsquoShu Sheng Peng Morsquo and lsquoXiao Xuersquo, were found to have only cyanidin glycoside and no anthocyanins, respectively. According to the chemical composition of anthocyanins, Xibei tree peony cultivars were classified into three phenotypes: (1) lsquoPn, Cyrsquo (33 cultivars); (2) lsquoPn, Pg>Cyrsquo (3 cultivars); and (3) lsquoPn, Cy>Pgrsquo (the remaining cultivars) (Wang et al. 2001a).

Floral flavones and flavonols

Twenty nine Xibei tree peony cultivars were found to contain three flavones (Ap, Lu, Ch) and three flavonols (Km, Qu, and Is) (Table 3, Wang et al. 2001b). Based on the kind of flavonoid in the plain part of petals, cultivars of Xibei tree peony are primarily divided into two specific phenotypes. One phenotype contains Km, Is, and Ap only (10 cultivars) and another all six flavonoids (29 cultivars). The former resembled Paeonia jishanensis and the latter resembled P. rockii (pink flowered) and P. potaninii (Wang et al. 2001b).

PCA and cluster analysis
The five eigenvalues (lambda i) were derived from PCA for the five factors (Z 1–Z 5), respectively (Table 4). The proportion (lambda i/Sgrlambda j) of each eigenvalue was computed and the cumulative proportion [(Sgrlambda i)/(Sgrlambda j)] of the five factors was found for a total of 100%. The pigments consisting of Pn3G5G, Cy3G, and Pn aglycone, the types of glycosides, as well as Km, greatly influenced the first factor, Z 1 (Table 5). Most of the flavonoid pigments were found to be relatively important for the second factor, Z 2, whereas the third factor, Z 3, was relatively dependent on Pg3G5G, Pg and TF. Pg3G and CI were found to dominate the fourth factor, Z 4.
Table 4  Eigenvalues, proportions and cumulative proportions obtained by principal component analysis, based on factors obtained from 20 pigment patterns

Factor

Eigenvalue, lambda i

Proportion, lambda i/(sumlambda j) (%)

Cumulative proportion, (sumlambda)/(sumlambda j) (%)

Z 1

39.567

56.59

56.59

Z 2

21.543

30.81

87.40

Z 3

4.600

6.58

93.98

Z 4

2.772

3.97

97.95

Z 5

1.437

2.06

100
Table 5  Eigenvectors obtained by PCA

Pigment pattern

Eigenvector

Z 1

Z 2

Z 3

Z 4

Z 5

Pn3G5G

0.7810

–0.4588

0.0131

0.1306

0.1151

Pn3G

–0.0383

0.5374

0.2421

–0.3597

–0.3210

Cy3G5G

0.5866

0.5519

0.0648

–0.2899

0.0397

Cy3G

–0.7481

0.4904

–0.1254

0.1245

0.2989

Pg3G5G

–0.3446

–0.4718

0.6383

–0.2012

0.4368

Pg3G

0.0813

0.0485

0.1348

0.6294

0.0342

Pn

0.8034

–0.3547

0.0669

0.0545

0.0461

Cy

–0.5605

0.7019

–0.1087

0.0241

0.3209

Pg

–0.3389

–0.4686

0.6486

–0.1553

0.4396

3G5G

0.8127

–0.3876

0.1660

0.0023

0.2113

3G

–0.7059

0.5943

–0.0557

0.0386

0.1993

Km

–0.7077

–0.4500

0.3045

–0.0213

–0.2871

Qu

0.4909

0.6548

0.3359

0.1546

–0.1023

Is

0.6651

0.4670

0.2213

0.3381

–0.0538

Ap

–0.2798

–0.5103

–0.5654

–0.1879

0.1441

Lu

0.5493

0.5302

–0.1927

–0.1678

0.3597

Ch

0.6425

0.1221

–0.3650

0.1894

0.4073

TA

0.0609

0.6193

0.0907

–0.5024

0.0478

TF

0.1575

0.4801

0.6480

0.1627

–0.0337

CI

–0.6411

0.1040

0.0231

0.6607

0.0420
Abbreviations: Pn3G5G Peonidin 3,5-di-O-glucoside; Pn3G peonidin 3-O-glucoside; Cy3G5G cyanidin 3,5-di-O-glucoside; Cy3G cyanidin 3-O-glucoside; Pg3G5G pelargonidin 3,5-di-O-glucoside; Pg3G pelargonidin 3-O-glucoside; Pn peonidin; Cy cyanidin; Pg pelargonidin; 3G5G 3,5-di-O-glucoside; 3G 3-O-glucoside; Km kaempferol, Qu quercetin, Is isorhamnetin, Ap apigenin, Lu luteolin, Ch chrysoeriol; TA total anthocyanins; TF total flavonols; CI copigment index
Two clusters, A and B, were derived using the program PROC TREE (Fig. 4). Cluster A included 11 cultivars with two species, Paeonia jishanensis and P. rockii (white flowered), and cluster B was composed of 28 cultivars with six species, P. rockii (pink flowered), P. ostii, P. qiui, P. decomposita, P. delavayi and P. potaninii. Comparatively, cluster A was based on the lowest constitution of yellow pigments such as Qu, Lu, and Ch, whereas cluster B was based on all six kinds of yellow pigments (Fig. 4).
MediaObjects/s10265-003-0130-6flb4.gif
Fig. 4  Dendrogram of 39 Xibei tree peony cultivars and 7 species (8 accessions) generated from similarity matrices obtained by Wardrsquos minimum variance cluster analysis in which semipartial R2 values were computed from Euclidean distance based on principal component analysis of 20 pigment patterns
Discussion

It has been believed that Xibei tree peony is derived from two species, Paeonia rockii and P. jishanensis (Qin 1997). Li and coworkers (Li et al. 1998; Li 1999) arranged the establishment of tree peony between cultivars and species based on morphological characters and concluded that Xibei cultivars originated from these two species as well as Zhongyuan cultivars. As a matter of fact, 11 cultivars made a subcluster (cluster A) with these species, but the other 28 cultivars did not. In cluster B, we found that 17 Xibei tree peony cultivars were closely related to pink flowered P. rockii. Since we presumed the accession of pink flowered P. rockii to be a hybrid between P. rockii (white flowered) and P. potaninii (Hong et al. 1992; Wang et al. 2001b), the result was well consistent with our former presumption.

Li and coworkers (Li et al. 1998; Li 1999) also pointed out that Zhongyuan cultivars, the representative of Chinese Mudan, are presumed to be the progeny of Paeonia ostii, P. rockii and P. jishanensis. However, it was difficult for them to distinguish among cultivars originating from only one or several species, as accessions occurred by lost mutation, and cultivars by artificial and geographical contamination. Thus, the inter-relationship between the two cultivar groups, Zhongyuan and Xibei, was thought to be much more complicated than their morphological appearance.

It was complicating that seven Xibei cultivars made a subcluster with Paeonia delavayi and four cultivars with P. potaninii in cluster B. However, it is hard to believe that the Xibei cultivar was derived from these two Delavayanae species, historically or geographically. We might suggest that these 11 cultivars are the progeny of Zhongyuan cultivars, which were probably related to Delavayanae species. Thus, Xibei tree peony originates mainly from P. rockii, and several cultivars occurred by crossing P. jishanensis and/or Zhongyuan cultivars. Although we are attempting to make a chemotaxonomic tree for Zhongyuan cultivars and original species, this suggestion agrees with the former postulate (Li et al. 1998; Li 1999).

Acknowledgements  The authors are grateful to Mr. De-Zhong Chen and Miss Fu-Hui Chen, of the Yuzhong Peace Peony Garden, Gansu Province, China for plant materials, and much help and guidance. We also thank Dr. Fang-Yun Cheng of the College of Landscape Architecture, Beijing Forest University, China for valuable suggestion and cooperation.

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