Flower pigment composition of Crocus species and cultivars used for a chemotaxonomic investigation

Biochemical Systematics and Ecology 30 (2002) 763–791 www.elsevier.com/locate/biochemsyseco

Flower pigment composition of Crocus species and cultivars used for a chemotaxonomic investigation R. Nørbæk a,∗, K. Brandt a, J.K. Nielsen b, M. Ørgaard c, N. Jacobsen c a

Danish Institute of Agricultural Sciences, Department of Horticulture, Kirstinebjergvej 10, DK-5792 A˚rslev, Denmark b Chemistry Department, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark c Department of Ecology, Botanical Section, Royal Veterinary and Agricultural University, Rolighedsvej 21, DK-1958 Frederiksberg C, Denmark Received 5 July 2001; accepted 28 November 2001

Abstract A survey of floral anthocyanins and other flavonoids by analytical high-performance liquid chromatography (HPLC) was performed among 70 species and subspecies, 43 cultivars and six artificial hybrids of Crocus and the results were compared with taxonomical delimitations established by Mathew (The Crocus. B.T. Batsford Ltd, London, 1982). Nine anthocyanins were detected. The Crocus species and cultivars were placed into seven chemotypes according to their contents of 3,7-di-O-, 3,5-di-O-glucosides or 3-O-rutinosides of delphinidin and petunidin and to the presence of 3,7-di-O-malonyl-glucosides of petunidin and malvidin and delphinidin 3-O-glucoside-5-O-malonylglucoside. These malonated anthocyanins have only been found in Crocus and may be characteristic for this genus. The same 18 flavonoids were detected in every taxon. However, quantitative differences were noted and four chemotypes of Crocus were defined by their major contents of flavonoids. Six of the flavonoids appear to be unique for Crocus. The anthocyanin/flavonoid patterns of some of the taxa provide a valuable supplement to the taxonomy based on morphological and cytological patterns. Most chemotypes were represented in several series but the chemical data were useful in distinguishing different species. For all series except Series h the chemical data were very similar for all subspecies or Corresponding author. Tel.: +45-63904302; fax +45-63904395. E-mail address: [email protected] (R. Nørbæk).

∗

0305-1978/02/$ - see front matter  2002 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 5 - 1 9 7 8 ( 0 2 ) 0 0 0 2 0 - 0

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accessions within a species, and chemotypes within a series were more similar than between series. However for six species, the analyses suggest that they should be further investigated using other methods, to evaluate their relations to other series.  2002 Elsevier Science Ltd. All rights reserved. Keywords: Crocus; Iridaceae; Flavonoids; Anthocyanins; HPLC; Chemotaxonomy

1. Introduction The history and taxonomy of Crocus has been dealt with in the two comprehensive monographs by Maw (1886) and Mathew (1982). Mathew’s (1982) monograph on the genus Crocus lists 80 species, and since then, half a dozen additional species and subspecies have been described. However, the ranking of some of the newly described taxa have been questioned, so about 83 species and additional three subspecies are generally accepted today, depending on taxonomical opinion. The genus Crocus is divided into two subgenera, viz. subgenus Crocus comprising all species except one, viz. C. banaticus which is the sole member of the subgenus Crociris. The subgenus Crocus is further divided into two sections viz. section Crocus and section Nudiscapus, each again divided into Series a–f and g–o, respectively. This subdivision of the genus is based on morphological as well as cytological characters. Although there are instances where Mathew’s classification does not seem quite adequate, it is difficult to suggest and justify reasonable changes i.e. you may question the present position of a given species, but it becomes difficult to suggest where should it then be placed, and you may easily end up creating e.g. new monotypic series. In some species the taxonomy is rather complicated, since the various classification characters, i.e. distribution pattern, habitat, various morphological traits and cytological data, contribute confusing, non-correlating data to the problem of systematic and phylogenetic grouping. So additional independent characters will be useful to supplement existing characters. More than 100 cultivars of Crocus are known today. They are derived from selection within and hybridisation between relatively few species. Series h comprises the annulate species (see also Series n), including the many subspecies of C. biflorus. This series is characterised by a smooth tunica, which for most species also includes rings at the base and a circular basal plate; the style is three-partitate. The colour of the flowers range from yellow to purple and blue, plain in colour to variously suffused spotted striped patterns, characteristic for each species or subspecies. However, there is some variation, also within populations with regards to colour and especially markings, as well as albino mutations that occur at low frequencies. In the Crocus chrysanthus–C. biflorus cultivars, it is difficult to say to what extent they are indeed hybrids between C. chrysanthus and C. biflorus as the name implies (Jacobsen et al., 1997). The aim of the present study was to provide new characters and evaluate whether they were useful to resolve ambiguities in the existing classification scheme.

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Especially the C. chrysanthus–C. biflorus cultivars have been studied to find out which cultivars are most likely selections of either of the two species or hybrids between them (Jacobsen et al., 1997). From previous studies it appeared that anthocyanins and carotenoids are the most important pigments determining the colour of Crocus flowers (Harborne and Williams, 1984; Nørbæk and Kondo, 1998, 1999a). The colour variation caused by anthocyanins in the flowers of Crocus ranges from purple or brownish markings or stripes on the outer perianth segments to uniformly coloured lilac mauve, or blue flowers. In the following, carotenoid chemistry will not be discussed with respect to the pigment composition. Recently nine anthocyanins have been reported as responsible for the cyanic colours (lilac, mauve and blue) of Crocus flowers. They were identified by modern NMR techniques as 3,7-di-O-b-glucosides, 3,5-di-O-b-glucosides and 3-O-b-rutinosides of delphinidin and petunidin, respectively, and delphinidin 3-O-b-glucoside-5O-(6-O-malonyl-b-glucoside) and 3-O-(6-O-malonyl-b-glucoside)-7-O-(6-O-malonyl-b-glucoside) of petunidin and malvidin (Nørbæk and Kondo, 1998, 1999a). The colourless flavonoids in Crocus flowers have been reported as 3-O-a-(2-Ob-glucosyl)rhamnoside-7-O-b-glucosides, 3-O-a-(2-O-b-glucosyl)rhamnosides, 3-Ob-(2-O-a-rhamnosyl)glucosides, 3-O-b-sophorosides, 3,4⬘-di-O-b-glucosides of different flavonols. Also included were kaempferol 3-O-a-(2-O-b-glucosyl)rhamnoside7-O-b-glucosides acylated with malonic acid or acetic acid in the OH-6 position of the 7-glucosides, kaempferol 3-O-a-(2,3-di-O-b-glucosyl)rhamnoside, kaempferol 3O-b-glucoside and 7-O-b-glucosides of apigenin and dihydrokaempferol (Nørbæk et al., 1999; Nørbæk and Kondo (1999b). This paper is concerned with the analytical contents of the cyanic flower pigments and other flavonoids in 123 taxa of Crocus in order to determine their distribution and usefulness as chemotaxonomical markers. Their retention characteristics in a standard HPLC system are given and the results are compared with morphological and cytological characters (Mathew, 1982; Ørgaard and Heslop-Harrison, 1994; Ørgaard et al., 1995a,b; Jacobsen et al., 1997).

2. Materials and methods 2.1. Plant material Flower pigments in 70 species, 43 cultivars and six produced hybrids of Crocus have been analysed. All of the artificial Crocus hybrids and most of the species and cultivars were grown at The Royal Veterinary and Agricultural University, Copenhagen. P-numbers were obtained from the Botanical Garden, University of Copenhagen (Table 6). The fresh perianth segments were frozen (⫺80°), then freeze-dried and analysed within six months.

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2.2. Analysis of perianth segments For analytical HPLC, about 1 g of the freeze-dried perianth segments were extracted with 13 ml 50% aq. CH3CN containing 3.0% TFA and after filtration the extracts were analysed by ODS HPLC (4.6φ × 250 mm, Develosil ODS-HG-5, Nomura Chemicals) at 35°, detection on a 3D diode-array detector 280–530 nm. 2.2.1. Identification of anthocyanins Two gradient systems were used to verify the presence of the anthocyanins. One was a linear gradient elution with a flow rate of 1 ml min ⫺1 for 30 min using from 0 to 30% aq. CH3CN containing 0.5% TFA. The second elution profile was as follows: 0 min 16% B, 3 min 38% B, 10 min 44% B, 20 min 50% B, 25 min 67% B, 40–50 min 100% B using solvent A (H2O–TFA, 99:1) and solvent B (CH3CN–H2O– TFA, 60:140:1), with a flow rate of 1.5 ml min⫺1. The anthocyanins were identified by their retention times and UV-spectra (Table 1) in accordance with Nørbæk and Kondo (1998, 1999a). The limit for detection at 530 nm was 1.2 µM for each anthocyanin in the extract, and injection volume was 50 µl. 2.2.2. Identification of flavonoids The flavonoids from the extracts were chromatographed using the second elution profile mentioned earlier. Detection was at 360 nm with injection volume 30 µl. The flavonoids were identified by their retention times and UV-spectra (Table 1) (Nørbæk et al., 1999; Nørbæk and Kondo, 1999b). In earlier work the anthocyanins and flavonoids have been identified by fast atom bombardment mass spectrometry (FAB-MS) as well as 1D and 2D nuclear magnetic resonance (NMR) (Nørbæk and Kondo, 1998; Nørbæk and Kondo, 1999a,b; Nørbæk

Table 1 Analytical HPLC retention times and lmax of anthocyanins and flavonoids isolated from perianth segments of Crocus Systematic names Anthocyanins A1 A2 A3 A4 A5 A6 A7 A8 A9

Delphinidin 3,7-di-O-b-glucoside Petunidin 3,7-di-O-b-glucoside Delphinidin 3,5-di-O-b-glucoside Petunidin 3,5-di-O-b-glucoside Delphinidin 3-O-b-rutinoside Petunidin 3-O-b-rutinoside Delphinidin 3-O-b-glucoside-5-O-b-(6-Omalonyl) glucoside Petunidin 3,7-di-O-b-(6-O-malonyl) glucoside Malvidin 3,7-di-O-b-(6-O-malonyl) glucoside

lmax (nm)

Rt (min)

5.7 8.2 9.2 12.4 13.5 16.6 14.3

280 279 269 269 282 275 271

537 537 538 537 515 542 538

16.1

269 536

17.5

278 536

(continued on next page)

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767

Table 1 (continued) Systematic names Flavonoids F10 F11

lmax (nm)

Rt (min)

F14

Dihydrokaempferol 7-O-b-glucoside Myricetin 3-O-a-(2-O-b-glucosyl)rhamnoside-7-O-b-glucoside Quercetin 3-O-a-(2-O-b-glucosyl)rhamnoside-7-O-b-glucoside Kaempferol 3-O-a-(2-O-b-glucosyl)rhamnoside-7-O-b-glucoside Quercetin 3-O-b-sophoroside

15.6

F15

Quercetin 3,4⬘-di-O-b-glucoside

17.0

F16

kaempferol 3,4⬘-di-O-b-glucoside

19.0

F17

Isorhamnetin 3,4⬘-di-O-b-glucoside

21.6

F18

Kaempferol 3-O-b-sophoroside

21.6

F19

Kaempferol 3-O-b-(2-O-a-rhamnosyl)glucoside Isorhamnetin 3-O-b-(2-O-a-rhamnosyl)glucoside Kaempferol 3-O-a-(2-O-b-glucosyl)rhamnoside-7-O-b-(6-O-malonyl)glucoside Kaempferol 3-O-a-(2,3-di-O-b-glucosyl) rhamnoside Kaempferol 3-O-a-(2-O-b-glucosyl) rhamnoside-7-O-b-(6-O-acetyl)glucoside Apigenin 7-O-b-glucoside

24.7

F12 F13

F20 F21 F22 F23 F24 F25

8.4 9.7

284, 335 sh 295, 358

12.2

255, 268 sh, 301 sh, 352 265, 310 sh, 345 257, 269 sh, 299 sh, 362 267, 301 sh, 354 265, 302 sh, 317 sh, 342 268, 300 sh, 351 267, 300 sh, 350 265, 295 sh, 317 sh, 347 253, 265 sh, 293 sh, 360 266, 301 sh, 315 sh, 345 268, 315 sh, 347 265, 290 sh, 315 sh, 345 270, 323 sh, 425 268, 315 sh, 347 270 sh, 299 sh, 363 266, 301 sh, 350

15.0

25.2 27.7 29.5 30.2 30.7 31.9

F26

Kaempferol 3-O-a-(2-O-b-glucosyl)rhamnoside Quercetin 3-O-b-glucosidea

F27

Kaempferol 3-O-b-glucoside

44.8

38.2

The structures were also determined by FAB-MS, 1D and 2D NMR spectroscopy (Nørbæk and Kondo, 1998; Nørbæk and Kondo, 1999a, b; Nørbæk et al., 1999). a only identified by co-chromatography and UV (in accordance with Markham, 1982).

et al., 1999). Quercetin 3-O-glucoside was only identified by co-chromatography and UV. 2.2.3. Data processing On the HPLC chromatograms for each genotype, the peak area of each anthocyanin or flavonoid was expressed as a percentage of the sum of the areas of all

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anthocyanin or flavonoid peaks, respectively. The ratings used in Tables 2 and 3 indicate the relatively percentages of anthocyanin as +++ correspond to ⬎55%; ++ correspond to ⬎30%; + correspond to ⬎10% and ± correspond to ⬍5%. The chemotypes (1–4) were defined by the contents of A5 and A6 (1), A3 and A4 (2), A1–A4 (3), A1–A6 (4). The chemotypes named with an additional m contained anthocyanins from chemotypes 1, 2 or 4 in combination with the malonated anthocyanins A7–A9 (see Table 1). The major enzymatic reactions involved in the flavonoid biosynthesis were categorised from the presence of flavonoids with corresponding glycosylation patterns. Based on these, four flavonoid groups (I–IV) were defined by their major composition of F19, F20 (I) (⬎20%); F14, F18 (II) (⬎20%); F15–F17 (III) (⬎20%) and F11–F13, F21–F23, F25 (IV) (⬎40%) (see Table 1). The relative amounts are shown in parentheses. To test if one or two enzymes were involved in 2-glucosylation with rhamnose or glucose, the ratio: percentages of 3-O-b-sophorosides (F14+F18)/(percentages of 3-O-a-(2-O-b-glucosyl)rhamnosides (F11+F12+F13+F21+F22+F23+F25)+3-O-βsophorosides (F14+F18)) were calculated for every genotype (Fig 1). Similarly, the ratios: percentages of kaempferol 3-O-a-(2-O-b-glucosyl)rhamnoside7-O-b-(6-O-malonyl)glucoside (F21)/(percentages of kaempferol 3-O-a-(2-O-bglucosyl)rhamnoside-7-O-b-(6-O-malonyl)glucoside (F21)+kaempferol 3-O-a-(2-O-bglucosyl)rhamnoside-7-O-b-(6-O-acetyl)glucoside (F23)) were tested. 2.2.4. Distance analysis The chemotaxonomical results were based on distance analyses using PAUP∗4.0 (Phylogenetic Analysis using parsimony) (Forey et al., 1996).

3. Results and discussion In Fig. 2, Mathew’s classification is outlined with the characteristics of each subdivision included. The outline includes only a few characteristics, as otherwise it quickly would become much too detailed to be dealt with in an overview, and Mathew’s work must be consulted for further details. The HPLC retention characteristics and UV data of the detected pigments, nine anthocyanins and 18 flavonoids are given in Table 1. 3.1. The anthocyanins In Table 2 information about the anthocyanin contents in species of Crocus is shown and the chemotypes of anthocyanins in species of Series (h) are shown again in Table 3 together with their cultivars and hybrids. Taxa included in chemotype 1 are capable of making A5 and A6, which are very common pigments in plant species of Iridaceae (Ashtakala and Forward, 1971; Arisumi, 1974; Ishikura and Yamamoto, 1978; Ishikura, 1980; Williams et al., 1986;

A1

++

±

++

A2

Anthocyanins

C. etruscus C. baytopiorum ++ C. kosaninii C. vernus (scepusiensis 234) C. vernus (95-101) ± C. vernus (95-115) C. tommasinianus C. pelistericus C. minimus C. corsicus C. imperati C. versicolor C. longiflorus C. niveus C. serotinus ssp. clusii C. serotinus ssp. salzmanii C. nudiflorus C. medius C. kotschyanus C. vallicola ++ C. mathewii C. cartwrightianus ‘Albus’ C. pallasii-white (92-6) C. pallasii (90-12) C. pallasii HNL (91-99) C. asumaniae C. hadriaticus C. oreocreticus C. cartwrightianus C. sativus

Species

Table 2 Distribution of anthocyanins in Crocus taxa

++ ++

++ ++

++ +++ +++ ++ ++ + ++ ++ ++ + ++ + ++ + +

++

++ + ++ ++ ++ ++ ++ ++ ++ ± + ++ ++ ++

++

++

++ ++ ++ + + + + + ++ +++ +++ ++ ++ ++ +++

A4

A3

+ + ++ + + +

+

±

+ + + +

± + ±

+ ±

A5

+ ± ± ±

±

++

±

++ + + ± ±

± +

±

A6

±

+

+

±

A7

+ +

±

A8

± ±

+

A9

50 50 130 30 90 180 60 20 20 10 70 20 40 40 20 10 30 10 10 10 50 10 10 80 50 120

40 10 80 180

C ta

a a a b c c c c d d d d d d e e f f f f f f f f f f

a a a a

Series

(continued on next page)

4 4 4 4 4 4 4 4m 2 2 2 2 2m 4 2 3 1m 2 2 2 4 4 4 4 4m 4m

2 3 4 4

Chemotypes

R. Nørbæk et al. / Biochemical Systematics and Ecology 30 (2002) 763–791 769

A1

±

±

++

A2

Anthocyanins

C. ancyrensi C. cvijicii C. reticulatus ssp. hittiticus C. reticulatus ssp. reticulatus C. gargaricus C. robertianus C. sieberi ssp. nivalis C. sieberi ssp. sieberi C. sieberi ssp. sublimis C. veluchensis + C. abantensis C. angustifolius C. angustifolius ‘Minor’ ± C. cancellatus ssp. lycius C. cancellatus ssp. mazziaricus C. sieberi ssp. atticus C. reticulatus × C. angustifolius C. chrysanthus (92-74) C. chrysanthus (P 19925264) C. danfordiae C. danfordiae -white/blue C. biflorus ssp. melantherus C. biflorus ssp. stridii (HNBJ 6457) C. biflorus ssp. stridii

Species

Table 2 (continued)

++

+ ± + ±

+ +++ +++ ++ +++ ++

A3

++ +

+ ++ + + ± + + + +

+++

A4

+ ++ ++ ++ + + + + +

++ ++ +++ +++ +++ +++ +++

++ + ± ++ ++

+++ ++ +

A6

± +

++ ++ +++ + ++

++ +++

+++

A5

±

A7

±

A8

A9

40

20 10 20 40

30 10

20 20

10 10 10 50 40 30 40 70 30 10 10

10 10 80 80

C ta

1

1 1 1 1

1 1

h

h h h h

h h

g g × g

g g g g g g g g g g g

g g g g

Series

(continued on next page)

4 4m

2 2 2 2 2 3 4 4 4 4 4

1 1 1 1

Chemotypes

770 R. Nørbæk et al. / Biochemical Systematics and Ecology 30 (2002) 763–791

A1

+ + ± ++ ++ + ++ +

+

±

+ ± +

± + +

±

±

±

+

+

±

A4

+ ++ ++ + ± +

A3

+++ ++ ++ ±

A2

Anthocyanins

C. biflorus ssp. nubigena (92-44) C. biflorus ssp. nubigena (HNL 9448) C.biflorus ssp. nubigena (HNL 6637) C. biflorus ssp. pseudonubigena C. leichtlinii C. adanensis C. biflorus ssp. weldenii C. aerius C. biflorus ssp. taurii ± C. biflorus ssp. crewei C. biflorus ssp. adamii ‘Serevan’ C. biflorus ssp. alexandrii (DJJG 99-9) C. biflorus ssp. isauricus C. biflorus ssp. pulchricolor C. biflorus ssp. punctatus C. korolkowii C. alatavicus C. flavus C. olivieri C. vitellinus C. candidus C. graveolens C. antalyensis ± C. ‘Stellaris’ ± C. ‘Golden Yellow’

Species

Table 2 (continued)

+ ++ + +

++ + +

++ + +++ ± + ++ +++ ++

++ + + ++ +++ +++ +++ ++ ++ ++ + + ++ ++ ±

+

+ + +

±

±

+

±

±

+

A7

++ ++ + +

+ ++ ++ ++ +

+

+

++

++

A6

A5

±

+ ±

±

++

±

+

+

A8

+

± + ±

±

±

++

±

±

±

A9

60 30 20 30 70 10 10 30 10 10 50 70 10

70

10 30 10 20 40 120 80

20

40

40

50

C ta

i j j j j j j g × j g × j

h h h i

h

h h h h h h h

h

h

h

h

Series

(continued on next page)

4m 4m 4m 1 1 1 1 1 4 4 4m 4 4m

4m

1m 2 2 4 4 4 4m

1m

1m

1m

1m

Chemotypes

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carpetanus fleicheri pulchellus ‘Albus’ speciosus ‘Albus’ pulchellus pulchellus ‘Zephyr’ speciosus laevigatus boryi banaticus

A1

A2

Anthocyanins A4 ++

+ + ± ±

A3 +

± ±

+

++ ++ +++ ± + + +++

A5

+++

++ ++ ++ + +++ +++ + +

A6

±

A7

+

A8

++

A9 10 10 10 10 20 10 70 20 0 50

C ta

4

4 1 1 1 4 4 4m 1

Chemotypes

l m n n n n n o o Crociris

Series

Classification in series in accordance with Mathew (1982) (Table 6) and distance analysis (see Materials and methods). Key. Anthocyanin structures of A1–A9 see Table 1. Following compounds are included in the respective chemotypes; 1: A5 and A8; 2: A3 and A4; 3: A1–A4 ; 4: A1–A6 and additional m: A7–A9. Rating of anthocyanins in relative concentration on HPLC; +++: high, ++: intermediate, +: low, ±: trace. a Approximate total anthocyanin concentration in µM. Ct was calculated from the HPLC chromatograms by using the extinction coefficient of malvidin 3,5diglucoside (log e ⫽ 4.58) (Wrolstad, 1993).

C. C. C. C. C. C. C. C. C. C.

Species

Table 2 (continued)

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773

Table 3 Distribution of anthocyanins in Crocus Series Biflori (h), their cultivars, and hybrids Cultivars and artificial hybrids

A1 A2 A3 C. danfordiae-yellow C. danfordiae-white/blue C. chrysanthus (P 19925264) C. chrysanthus (92-74) C. biflorus ‘Major’ C. biflorus ssp. melantherus C. biflorus ssp. stridii (HNBJ 6457) C. biflorus ssp. stridii C. ‘Ard Schenk’ C. ‘Blue Bird’ C. ‘Creme Beauty’ C. ‘Elegance’ C. ‘Eye-catcher’ C. ‘Fairy’ C. ‘Fuscotinctus’ C. ‘Gipsy Girl’ C. ‘Harlequin’ C. ‘Herald’ C. ‘Jeannine’ C. ‘Miss Vain’ C. ‘Mrs. Moon’ C. ‘Romance’ C. ‘Saturnus’ C. ‘Snowbunting’ C. ‘Spotlight’ C. ‘Sulphur’ C. ‘Warley’ C. biflorus ssp. nubigena C. biflorus ssp. nubigena (HNL 9448) C.biflorus ssp. nubigena (HNL 6637)

Cta

Chemotypes

+++ + +++ + ++ ++

20 10 20

1 1 1

++ ++ +++ + +++ +

30 120 20

1 1 1

+++ +

40

1

+++ +++ +++ +++ +++ +++ +++ +++ ++ +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ ++

+ + +

+ ±

+ +

± ±

40 10 30 10 20 170 20 20 10 30 60 20 10 10 10 10 10 20 10 20 50 40

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1m 1m

+

++

±

±

±

40

1m

Anthocyanins

A4 A5

A6

A7 A8 A9

+ + + + + ++ + ++ +

+ +

(continued on next page)

Yabuya, 1987; Yabuya, 1991). The species in chemotype 2 and 3 are not so distantly separated, since they are only distinguished by the position of di-glucosylation. In most cases subspecies appear in the same anthocyanin chemotypes, i.e. subspecies of C. reticulatus (Series g) in chemotype 1, C. serotinus (Series d) in chemotype 2, C. cancellatus (Series g) in chemotype 4, while others, i.e. C. sieberi (Series g) appears with subspecies in chemotype 2 and 4 and C. biflorus (Series h) with sub-

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Table 3 (continued) Cultivars and artificial hybrids

Cta

Chemotypes

20

1m

10 40 20 30 10 20 70 40 10

1m 1m 1m 1m 1m 1m 1m 1m 1m

40

1m

±

30 20 20 120 40 40 80

2 2 4 4 4 4 4m

±

70

4m

+ ± ++ + ± +

60 30 30

4m 4m 4m

20 30 20 40 20

4m 4m 4m 4m 4m

Anthocyanins

A1 A2 A3 C. biflorus ssp. pseudonubigena C. leichtlinii C. ‘Aubade’ C. ‘Blue Jay’ C. ‘Brassband’ C. ‘Ladykiller’ C. ‘Prinses Beatrix’ C. ‘Skyline’ C. ‘Spring Pearl’ C. chrysanthus × C. biflorus ‘Major’ C. biflorus ‘Major’ × C. chrysanthus C. adanensis C. biflorus ssp. weldenii C. aerius C. biflorus ssp. crewei C. biflorus ssp. tauri ± C. ‘Zenith’ C. biflorus ssp. adamii ‘Serevan’ C. biflorus ssp. alexandrii (DJJG 99-9) C. biflorus ssp. isauricus C. biflorus ‘Parkinsonii’ C. biflorus ssp. pulchricolor C. biflorus ssp. punctatus C. ‘Advance’ C. ‘Blue Pearl’ C. ‘Blue Peter’ C. ‘Brunette’

± ±

A4 A5

A6

A7 A8 A9

+++ +

+

± + + ++ ++ ++ + + ++

+ +++ ++ + ++ + + + ++

± + + + + + + ±

+ + + + ++ + + ± ±

++

+

±

±

++ ++

+++ + ++ ++ ++ ++ ± ± + ± + + +

+ +++ ++ ++ ++

+ ++ ++ +

±

+

++

+

±

± + +

++ + +

+ ± ++

+ + +

± ± + ± +

+ ± ++ ± +

+ ++ ++ ++ ++

++ + + + +

+ + + + + + ++

± ±

± ±

(continued on next page)

species in chemotypes 1, 2, 4, 1m and 4m. None of the taxonomic series correspond exactly to the anthocyanin chemotypes except where only one taxon is involved. For example Series a, with five species, is represented in three anthocyanin chemotypes. The anthocyanins cause the cyanic colours in flowers (Harborne, 1996) and modifications like malonylation of the attached glucoside units and hydroxylation or methoxylation on the B-ring of the anthocyanidins will cause a shift to more bluish hues (Asen and Griesbach, 1983; Asen, 1984; Arisumi et al., 1985; Cohen et al., 1985; Yabuya, 1991; Yabuya et al., 1994; Nørbæk et al., 1998).

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Table 3 (continued) Cultivars and artificial hybrids

Anthocyanins

A1 A2 A3 C. ‘Goldilocks’ C. ‘Prins Claus’ C. ‘Zwanenburg Bronze’ ± C. biflorus ‘Major’ x C. ± biflorus ssp. crewei C. chrysanthus x C. biflorus ssp. pulchricolor C. aerius x C. adanensis

± ±

± ± ± ±

±

A4 A5

A6

A7 A8 A9

± ± ± ±

+++ ++ +++ ++

± + + ++

+ + ± ±

± ±

±

±

++

+

++ +

±

+

+

++

+

++

Cta

Chemotypes

10 10 100 60

4m 4m 4m 4m

10

4m

20

4m

Key. Anthocyanin structures of A1–A9 see Table 1. Following compounds are included in the respective chemotypes; 1: A5 and A8; 2: A3 and A4; 3: A1–A4 ; 4: A1–A6 and additional m: A7–A9. Rating of anthocyanins in relative concentration on HPLC; +++: high, ++: intermediate, +: low, ±: trace. a Approximate total anthocyanin concentration in µM. Ct was calculated from the HPLC chromatograms by using the extinction coefficient of malvidin 3,5-diglucoside (log e ⫽ 4.58) (Wrolstad, 1993).

Fig. 1. Stereostructures of kaempferol 3-O-b-glucopyranoside and 3-O-a rhamnopyranoside, respectively. Two different 2-O-glucosyltransferases control the formation of flavonol 3-O-sophorosides (1) (2OGT1) and flavonol 3-O-(2-O-glucosyl)rhamnosides (2) (2OGT2), respectively. Since the non-normal data of percentages of (3-O-b-sophorosides (F14+F18)/(percentages 3-O-a-(2-O-b-glucosyl)rhamnosides (F11+F12+F13, F21+F22+F23+F25)+3-O-b sophorosides (F14+F18)) varied prominently from 0-80% in the flowers (Tables 4 and 5).

Correlations between anthocyanin type and natural selection for flower colour has been documented (Harborne, 1967) and as an example, an evolutionary trend towards blue flowers has been reflected in the frequency of blue-coloured species in temperate members of Labiatae, Polemoniaceae and Boraginaceae (Harborne, 1983). A selection pressure for flower colour may have influence on the contents of malonated anthocyanins (A7–A9) in Crocus (Table 1). It is likely that the presence of two genes with dominant alleles that promote methoxylation in the 2⬘ and 4⬘

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Fig. 2. An outline of the classification of the genus Crocus according to Mathew (1982) modified to include new taxa.

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777

positions of the chromophore, and malonylation on 6-OH position of the glucoside moieties, respectively, each make the perianth segments more blue (Nørbæk et al., 1998). The presence of a selection pressure was indicated by the frequent co-occurrence of the two characteristics. Out of 14 species containing malonated anthocyanins nine also contained malvidin (A9) (Table 2). Thus, the acylated anthocyanins A7– A9 included in chemotypes 1m, 2m and 4m cannot be considered as neutral markers, since these compounds may appear as a result of selection for blue colour hue. Malonated anthocyanins appear in many different series of Crocus (Series c, d, f, g, h, j, and n). It is likely that the ancestor of the genus would have been capable of producing methoxylated and malonated anthocyanins, but that these characters were lost in some species during evolution. This possible explanation is supported by the fact that acylated flavonoids and thus the relevant class of enzymes are found in every taxon of Crocus. It is possible that a mutation occurred so the specificity of substrate for the acyltransferase in Crocus evolved from including only flavonols to also including anthocyanins. However, it is likely that selection for colour does not have an affect on compounds A1–A6 (Table 1) because the difference among the glucosyl- and rutinosylmoieties do not affect the intensity of the blue perianth colour, so these compounds may have a considerable chemotaxonomic importance (Nørbæk et al., 1998). A3– A6 are widespread within the genus and therefore can be considered as ancestral compounds, while the not so frequently appearing A1 and A2 may have joined later. In all it seems that evolution of new enzymes have had little importance for the anthocyanin pattern within the genus, because most of the observed changes are based on loss of ability to produce certain compounds. 3.2. The flavonoids The taxa in Tables 4 and 5 have been arranged in order to be compared with Tables 2 and 3, respectively. Every taxon of Crocus contains 18 detected flavonoids. With regard to flavonoid aglycones, kaempferol dominates, as kaempferol glycosides constitute between 70 and 90% of the total contents of flavonoids in the flowers of the genus. The contents of quercetin glycosides varied from 5 to 10% in all taxa and glycosides of dihydrokaempferol, isorhamnetin, myricetin and apigenin were only minor components. Four groups of flavonoids were defined, reflecting the major composition of 14 flavonoids (see Materials and methods and Table 1). The remaining four flavonoids: 7-O-b-glucosides of dihydrokaempferol and apigenin and 3-O-b-glucosides of kaempferol and quercetin appeared as minor and major components, respectively. No considerable variation of the amounts of these compounds was found and they were not treated any further because of no chemotaxonomical value. In comparison to the anthocyanins the flavonoids present a simpler picture. The flavonols and flavones, although possessing a weak yellow colour as pure substances, often impart no visible colour to the tissues in which they are found (Harborne, 1996). However, the flavonols and flavones are potential co-pigments, and their sugar units may have importance for the interactions with anthocyanins. Co-pigments do

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Table 4 Distribution of major flavonoids in Crocus taxa Crocus species

Flavonoid groups I

C. etruscus C. baytopiorum C. kosaninii C. vernus (scepusiensis 234) C. vernus (95-101) C. vernus (95-115) C. tommasinianus C. pelistericus C. minimus C. corsicus C. imperati C. versicolor C. longiflorus C. niveus C. serotinus ssp. clusii C. serotinus ssp. salzmanii C. nudiflorus C. medius C. kotschyanus C. vallicola C. mathewii C. cartwrightianus ‘Albus’ C. pallasii white (92-6) C. pallasii (90-12) C. pallasii (HNL 9199) C. asumaniae C. hadriaticus C. oreocreticus C. cartwrightianus C. sativus C. ancyrensis C. cvijicii C. reticulatus ssp. hittiticus C. reticulatus ssp. reticulatus C. gargaricus C. robertianus

II

Series III

IV

A

+ + + +

+ + +

0.24 0.007 0.33 0.57

a a a a

+

+ + +

+ + + + + + + + + + +

0.20 0.090 0.43 0.15 0.17 0.53 0.21 0.09 0.17 0.046 0.083

a a a b c c c c d d d

+

+

+

0.053

d

+ + +

0.082 0.21 0.058 0.007 0.089 0.042

d d e e f f

+

+ + + +

+

+ + +

+ +

+

+

+

0.004

f

+ +

+ +

0.009 0.063

f f

+ + +

+ + + + + + + +

0.019 0.013 0.55 0.50 0.39 0.018 0.005 0.31

f f f f f g g g

+

+

0.044

g

+

0.016 0.035

g g

+

+

+

+

+

(continued on next page)

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779

Table 4 (continued) C. sieberi ssp. nivalis C. sieberi ssp. sieberi C. sieberi ssp. sublimis C. veluchensis C. abantensis C. angustifolius C. angustifolius ‘Minor’ C. cancellatus ssp. lycius C. cancellatus ssp. mazziaricus C. sieberi ssp. atticus C. reticulatus × C. angustifolius C. chrysanthus (9274) C. chrysanthus (P 1992-5264) C. danfordiae C. danfordiaewhite/blue C. biflorus ssp. melantherus C. biflorus ssp. stridii (HNBJ 6457) C. biflorus ssp. stridii C. biflorus ssp. nubigena (92-44) C. biflorus ssp. nubigena (HNL 9448) C. biflorus ssp. nubigena (HNL 6637) C. biflorus ssp. pseudonubigena C. leichtlinii C. adanensis C. biflorus ssp. weldenii C. aerius C. biflorus ssp. tauri

+

+

0.034

g

+

+

0.11

g

+

+

0.11

g

+ +

+ + +

0.14 0.14 0.020 0.16

g g g g

+

+

0.038

g

+

+

0.014

g

0.048

g

+ + +

+

+ +

+

0.070

gXg

+

+

+

0.003

h

+

+

+

0.003

h

+ +

0.061 0.012

h h

+

0.070

h

+

0.21

h

+

0.18

h

+

0.28

h

+

0.30

h

+

0.24

h

+

0.036

h

+

0.055 0.058 0.086

h h h

0.11 0.20

h h

+

+

+

+

+

+

(continued on next page)

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Table 4 (continued) Crocus species

Flavonoid groups I

C. biflorus ssp. crewei C. biflorus ssp. adamii ‘Serevan’ C. biflorus ssp. alexandrii (DJJG 999) C. biflorus ssp. isauricus C. biflorus ssp. pulchricolor C. biflorus ssp. punctatus C. korolkowii C. alatavicus C. flavus C. olivieri C. vitellinus C. candidus C. graveolens C. antalyensis C. ‘Stellaris’ C. ‘Golden Yellow’ C. carpetanus C. fleicheri C. pulchellus ‘Albus’ C. speciosus ‘Albus’ C. pulchellus C. pulchellus ‘Zephyr’ C. speciosus C. laevigatus C. boryi C. banaticus

II

+

Series III

0.18

h

+

0.51

h

+

0.34

h

+

0.24

h

+

0.27

h

+

0.081

h i i j j j j j j g × j g × j l m n

+

+ + + + +

+

+ +

0.19 0.20 0.005 0.016 0.12 0.004 0.020 0.041 0.004 0.004 0.025 0.019 0.017

+ + +

+ + +

0.006 0.24 0.011

n n n

+ + +

+

0.16 0.10 0 0.12

n o o Crociris

+

+

+

A

+

+

+

IV

+ + + + + +

+ + + + +

+

Classification in accordance with Mathew (1982) and distance analysis (see Materials and methods).Key. A: absorption of anthocyanin at 535 nm divided by the absorption of flavonoid at 360 nm. Grouping by contents of flavonoid structures F11–F23, F25 (see Table 1). Enzymes shown in parantheses. 1: ⬎20% F19, F20 (FGT, 2ORT); 2: ⬎20% F14, F18 (FGT, 2OGT1); 3: ⬎20% F15-F17 (FGT, F4⬘OG); 4: ⬎50% F11–F13, F21–F23, F25, (FRT, F7OG, 2OGT2, 3OGT, AT). FGT, Flavonoid 3-O-glucosyltransferase; 2ORT, Flavonoid 2-O-rhamnosyltransferase; 2OGT1, Flavonoid 2-O-glucosyltransferase (3-Osophoroside); F4⬘OG, Flavonoid 4⬘-O-glucosyltransferase; FRT, Flavonoid 3-O-rhamnosyltransferase; F7OG, Flavonoid 7-O-glucosyltransferase; 2OGT2, Flavonoid 2-O-glucosyltransferase (3-O-(2-Oglucosyl)rhamnoside); 3OGT, Flavonoid 3-O-glucosyltransferase; AT, acyltransferase. The minor components; 7-O-glucosides of dihydro-kaempferol (F10) and apigenin (F24) and and the major components; 3-O-glucosides of quercetin (F26) and kaempferol (F27) were not included in the grouping.

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Table 5 Grouping in accordance to the major composition of flavonoids in Crocus Series (h), their cultivars and hybrids Cultivars

Flavonoid groups I

C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C.

danfordiae-yellow danfordiae-white/blue chrysanthus (P 1992-5264) chrysanthus (92-74) biflorus ‘Major’ biflorus ssp. melantherus biflorus ssp. stridii (HNBJ 6457) biflorus ssp. stridii ‘Ard Schenk’ ‘Blue Bird’ ‘Creme Beauty’ ‘Elegance’ ‘Eye-catcher’ ‘Fairy’ ‘Fuscotinctus’ ‘Gipsy Girl’ ‘Harlequin’ ‘Herald’ ‘Jeannine’ ‘Miss Vain’ ‘Mrs. Moon’ ‘Romance’ ‘Saturnus’ ‘Snowbunting’ ‘Spotlight’ ‘Sulphur’ ‘Warley’ biflorus ssp. nubigena

II

+ +

III

+ + +

+

+

+ + + + + + + + + +

IV

A

+ + + + + + + + + + + + + + + + + + + + + + + + + + + +

0.061 0.012 0.004 0.003 0.28 0.070 0.21 0.18 0.0008 0.062 0.019 0.12 0.42 0.050 0.025 0.01 0.055 0.087 0.050 0.002 0.007 0.01 0.031 0.020 0.035 0.01 0.056 0.28 (continued on next page)

not by themselves significantly contribute to the colour, but they cause a bathochromic wavelength shift in the visible lmax of natural anthocyanins, which makes them appear bluer and increase absorptivity (Asen et al., 1972, 1975; Scheffeldt and Hrazdina, 1978; Kim and Fujieda, 1991). The absorption of anthocyanin divided by the absorption of flavonoids (A) are shown in Tables 4 and 5 for every taxa, since in earlier studies a linear relation with colour hue was shown in the genus Alstroemeria (Nørbæk et al., 1998). However, within a series this relation (A) vary by a factor 100 and no systematic relation with anthocyanin chemotype was evident. Most of the flavonoids from Crocus are of relatively widespread occurrence in the plant kingdom. However, the discovery of F11–F13, F21–F23 and F25 is to our knowledge unique for Crocus and separates the genus from other relatives in the Iridaceae. On the other hand, since the chemotaxonomy of Liliales is rudimentary,

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Table 5 (continued) Cultivars

Flavonoid groups I

C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C.

biflorus ssp. nubigena (HNL 9448) biflorus ssp. nubigena (HNL 6637) biflorus ssp. pseudonubigena leichtlinii ‘Aubade’ ‘Blue Jay’ ‘Brassband’ ‘Ladykiller’ ‘Prinsses Beatrix’ ‘Skyline’ ‘Spring Pearl’ chrysanthus × C. biflorus ‘Major’ biflorus ‘Major’ × C. chrysanthus adanensis biflorus ssp. weldenii aerius biflorus ssp. creweii + biflorus ssp. tauri + ‘Zenith’ biflorus ssp. adamii ‘Serevan’ biflorus ssp. alexandrii (DJJG 99-9) biflorus ssp. isauricus biflorus ‘Parkinsonii’ biflorus ssp. pulchricolor biflorus ssp. punctatus ‘Advance’ ‘Blue Pearl’ ‘Blue Peter’ ‘Brunette’ ‘Goldilocks’ ‘Prins Claus’ ‘Zwanenburg Bronze’ biflorus ‘Major’ × C. biflorus ssp. crewei chrysanthus × C. biflorus ssp. pulchricolor aerius × C. adanensis

II

III

+ + +

+

+ + + +

+ + + + +

+ + + + +

IV

A

+ + +

0.30 0.24 0.036 0.055 0.076 0.036 0.053 0.023 0.087 0.035 0.060 0.089 0.067 0.058 0.086 0.11 0.18 0.20 0.081 0.51 0.34 0.24 0.24 0.27 0.081 0.050 0.23 0.022 0.034 0.002 0.069 0.21 0.23 0.041 0.25

+ + + + + + + + + + + + + + + + + + + + + + + + + + +

Key. A: absorption of anthocyanin at 535 nm divided by the absorption of flavonoid at 360 nm. Grouping by contents of flavonoid structures F11–F23, F25 (see Table 1). Enzymes shown in parantheses. 1: ⬎20% F19, F20 (FGT, 2ORT); 2: ⬎20% F14, F18 (FGT, 2OGT1); 3: ⬎20% F15–F17 (FGT, F4⬘OG); 4: ⬎50% F11–F13, F21–F23, F25, (FRT, F7OG, 2OGT2, 3OGT, AT). FGT, Flavonoid 3-O-glucosyltransferase; 2ORT, Flavonoid 2-O-rhamnosyltransferase; 2OGT1, Flavonoid 2-O-glucosyltransferase (3-Osophoroside); F4⬘OG, Flavonoid 4⬘-O-glucosyltransferase; FRT, Flavonoid 3-O-rhamnosyltransferase; F7OG, Flavonoid 7-O-glucosyltransferase; 2OGT2, Flavonoid 2-O-glucosyltransferase (3-O-(2-Oglucosyl)rhamnoside); 3OGT, Flavonoid 3-O-glucosyltransferase; AT, acyltransferase. The minor components; 7-O-glucosides of dihydro-kaempferol (F10) and apigenin (F24) and and the major components; 3-O-glucosides of quercetin (F26) and kaempferol (F27) were not included in the grouping.

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Table 6 Nomenclature and origin of plant material. Voucher specimen are being held at the Botanical Museum of Copenhagen (C) and referred to by the number in parentheses. N. Jacobsen determined the specimens C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C.

abantensis T. Baytop and B. Mathew (P 1993-5312) adanensis T. Baytop and B. Mathew (P 1993-5235) aerius Herbert (P1993-5313) alatavicus Semenov and Regel (C 107) ancyrensis (Herbert) Maw (C 32) angustifolius Weston (C 116) angustifolius Weston ‘Minor’ (C 247) antalyensis B. Mathew (92-16) asumaniae B. Mathew and T. Baytop (C 338) banaticus Gay (P 1992-5240) baytopiorum B. Mathew (P 1980-5094) biflorus Miller ssp. alexandrii (Velen.) B. Mathew (DJJG 99-9) biflorus Miller ssp. crewei (Hook.) B. Mathew (92-38) biflorus Miller ssp. isauricus (Bowles) B. Mathew (90-33) biflorus Miller ‘Major’ (C 40) biflorus Miller ssp. melantherus (Boiss. and Orph.) B. Mathew (93-26) biflorus Miller ssp. nubigena (Herbert) B. Mathew (92-44) biflorus Miller ssp. pseudonubigena B. Mathew (C 339) biflorus Miller ssp. pulchricolor (Herbert) B. Mathew (C 100) biflorus Miller ssp. punctatus B. Mathew (90-18) biflorus Miller ssp. adamii (Gay) B. Mathew ‘Serevan’ (C 158) biflorus Miler. ssp. tauri (Maw) B. Mathew (C 404) biflorus Miller ssp. weldenii (Hoppe and Furnrohr) B. Mathew (95-110) boryi Gay (P 25313) cancellatus Herbert ssp. lycius B. Mathew (92-7) cancellatus Herbert ssp. mazziaricus (Herbert) B. Mathew (93-27) candidus Clarke (P 1992-5261) carpetanus Boiss. and Reut. (P 1994-5410) cartwrightianus Herbert (C 236) carwrightianus Herbert ‘Albus’ (C 253) chrysanthus (Herbert) Herbert (P 1992-5264) chrysanthus (Herbert) Herbert (92-74) corsicus Maw (C 96) cvijicii Kosanin (93-49) danfordiae Maw—yellow (90-80) danfordiae Maw—white/blue (90-71) etruscus Parl. (C 410) flavus Weston (C 49) fleicheri Gay (90-27) gargaricus Herbert (P 1992-5270) graveolens Boiss. (P 1992-5272) hadriaticus Herbert (P 1979-5459) imperati Ten. (95-54) korolkowii Maw (C 170) kosaninii Pulevic (C 139) kotschyanus K. Koch (C 78) laevigatus Bory and Chaub. (94-20) leichtlinii Bowles (C 351) (continued on next page)

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Table 6 (continued) C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C.

longiflorus Raf. (P 1992-5293) mathewii Kerndorff and Pasche (92-4B) medius Balb. (C 84) minimus DC. (C 15) niveus Bowles (P 1992-5298) nudiflorus Smith (P 1992-5300) olivieri Gay (G 88-3-2) oreocreticus Burtt (94-26) pallasii Goldb. (90-12) pallasii Goldb.—white (92-6) pallasii Goldb. (HNL 91-99) pallasii Goldb. (90-35) pelistericus Pulevic (C 350) pulchellus Herbert ‘Albus’ (C 407) pulchellus Herbert (93-41) pulchellus Herbert ‘Zephyr’ (C 242) reticulatus Adams ssp. hittiticus (T. Baytop and B. Mathew) B. Mathew (C 259) reticulatus Adams ssp. reticulatus (C 149) robertianus C. Brickell (P 1986-5411) sativus L. (C 340) serotinus Salisb. ssp. clusii (Gay) B. Mathew (P 1992-5319) serotinus Salisb. ssp. salzmanii (Gay) B. Mathew (C 429) sieberi Gay ssp. atticus (Boiss. and Orph.) B. Mathew (93-4) sieberi Gay ssp. nivalis (Bory and Chaub.) B. Mathew (C 441) sieberi Gay ssp. sieberi (90-03-02) sieberi Gay ssp. sublimis (Herbert) B. Mathew (93-8) speciosus M. Bieb. ‘Albus’ (C 11) speciosus M. Bieb. (C 79) tommasinianus Herbert (C 413) vallicola Herbert (C 123) veluchensis Herbert (C s.n.) vernus (L.) Hill (95-101) vernus (L.) Hill (95-115c) vernus (L.) Hill-scepusiensis (C 234) versicolor Ker-Gawler (C 446) vitellinus Wahlenb. (P 1992-5340) ‘Advance’ (C 82) ‘Ard Schenk’ (C 165) ‘Aubade’ (C 22) ‘Blue Bird’ (C 30) ‘Blue Jay’ (C 391) ‘Blue Pearl’ (C 24) ‘Blue Peter’ (C 169) ‘Brassband’ (C 166) ‘Brunette’ (C 226) ‘Creme Beauty’ (C 31) ‘Elegance’ (C 168) ‘Eye-catcher’ (C 38) ‘Fairy’ (C 171) ‘Fuscotinctus’ (C 35) (continued on next page)

R. Nørbæk et al. / Biochemical Systematics and Ecology 30 (2002) 763–791

785

Table 6 (continued) C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C. C.

‘Gipsy Girl’(C 164) ‘Golden Yellow’ (C 67) ‘Goldilocks’ (C 167) ‘Harlequin’ (C 279) ‘Herald’ (C 163) ‘Jeannine’ (C 263) ‘Ladykiller’ (C 62) ‘Miss Vain’ (C 128) ‘Mrs. Moon’ (C 80c) ‘Parkinsonii’(C 94) ‘Prinses Beatrix’ (C 81) ‘Prins Claus’ (C 85) ‘Romance’ (C 205) ‘Saturnus’ (C 45) ‘Skyline’ (C 37) ‘Snow Bunting’ (C 161) ‘Spotlight’ (C 34b) ‘Spring Pearl’ (C 162) ‘Stellaris’ (C 246) ‘Sulphur’ (C 195) ‘Warley’ (C 70) ‘Zenith’ (C 160) ‘Zwanenburg Bronze’ (C 155) aerius × C. adanensis (CC 203) biflorus ‘Major’ × C. biflorus ssp. creweii (CC 518) biflorus ‘Major’ × C. chrysanthus (CC 197) chrysanthus × C. biflorus ‘Major’ (CC 537) chrysanthus × C. biflorus ssp. pulchricolor (CC 532) reticulatus × C. angustifolius (CC 67)

future phytochemical studies may reveal such flavonol glycosides in other genera of Iridaceae. Flavonoid group I–III all have high activity of a 3-O-glucosyltransferase (FGT), an enzyme earlier described in flowers (van Nigtevecht and van Brederode, 1975); Besson et al., 1979; Hrazdina, 1988; Ishikura and Yamamoto, 1990) (Tables 4 and 5). In addition, group I is characterised by high activity of a 2-O-rhamnosyltransferase (2ORT). Flavonoid group II has an active flavonoid 2-O-glucosyltransferase (2OGT1) (van Brederode and van Nigtevecht (1974)) and flavonoid group III represent taxa with high activity of a flavonoid 4⬘-O-glucosyltransferase (F4⬘OG) (Latchinian-Sadek and Ibrahim, 1991). Group IV is characterised by taxa having relatively high activity of five enzymes; a flavonoid 3-O-rhamnosyltransferase (FRT), a flavonoid 7-O-glucosyltransferase (F7OG), a flavonoid 2-O-glucosyltransferase (2OGT2), a flavonoid 3-O-glucosyltransferase (3OGT) and an acyltransferase (AT). The stereochemical differentiation between b-glucopyranoside and a-rhamnopyranoside is prominent and, therefore, different glucosyltransferases are probably needed for the 2-O-glucosylation (2OGT1, 2OGT2) (Fig. 1). This is supported by

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great variation of the relative amounts of flavonol 3-O-b-sophorosides (F14+F18) and 3-O-a-(2-O-b-glucosyl)rhamnosides (F11+F12+F13, F21+F22+F23+F25) ranging from 0 to 80% of all flavonoid 3-diglycosides. Since F22 is an end product, the only substrate for the glucosyltransferase responsible for the 3-O-glucosylation seems to be kaempferol 3-O-a-(2-O-bglucosyl)rhamnoside, because no flavonol 3-O-a-(3-O-b-glucosyl)rhamnosides were found in the Crocus flowers. Acylation of flavonoid metabolites is generally a late event in flavonoid biosynthesis (Harborne, 1996). An insignificant variation in the ratio of the co-existing F21 and F23 was found, so the same acyltransferase is probably catalysing both malonylation and acetylation (see Materials and methods). The results are consistent with the hypothesis that only one enzyme catalyses both reactions as has been shown earlier for similar enzymes (Fujiwara et al., 1998). From Tables 4 and 5 it appears that groups I–III are not evenly distributed among the taxa and, therefore, have a chemotaxonomic importance. However, group IV is dominant in almost all taxa, so this group has less importance for the chemosystematics within the genus. 3.3. The anthocyanins and other flavonoids in the Crocus species The survey of anthocyanins shows that in Series a, b, c, f and g chemotype 4 frequently appear (Table 2). However C. baytopiorum (Series a) is placed in chemotype 3 as well as C. vallicola (Series e). Also the flavonoid pattern of C. baytopiorum deviates from other members of Series A. While this does not by itself discredit earlier interpretations it indicates that the position of this species merits further investigation. Series b is only represented by C. pelistericus (the other: C. scardicus was not investigated here). It is noticable that C. pelistericus has similar anthocyanin and flavonoid patterns as most of members of Series a. In Series c the chemotaxonomic evidence is closely in accordance with Mathew (1982). The two non-white C. pallasii accessions and C. cartwrightianus (Series f) belong to chemotypes 2, 4 and 4m, respectively, while the white forms are in chemotype 2. The apparently simpler anthocyanin pattern in taxa regarded as white forms may be due to the physical limit of detection. That the anthocyanin pattern of C. sativus resembles that of C. cartwrightianus could indicate agreement with earlier proposals, i.e. C. sativus being a triploid of C. cartwrightianus. However, the flavonoid patterns do not support such an interpretation. A noteworthy characteristic is that C. mathewii differs from the rest of the taxa in Series f (Table 4), both for anthocyanins and flavonoids. In Series g C. sieberi is represented by its four subspecies, where the anthocyanin pattern of C. sieberi ssp. atticus deviates from the others. From the major flavonoid contents it is noteworthy that C. sieberi ssp. atticus again does not group together with other subspecies (Table 4). However, it is not explainable why C. sieberi ssp. atticus is so much different from the other C. sieberi subspecies. It is interesting that the morphologically similar C. veluchensis differs in both the anthocyanin and

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flavonoid pattern. This supports the opinion that C. veluchensis and C. sieberi are different species. Crocus medius deviates from other taxa in Series d, and two species; C. kotschyanus and C. vallicola differ in both anthocyanin and flavonoid contents regarding the results of Series e. Therefore, the chemical data do not by themselves support their inclusion in the same series (Tables 2 and 4). Series h is described separately in Section 3.4. Series i–o comprise chemotypes 1, 4 and 4m, especially supporting a close connection between i, j and m. Series i is represented by two species, C. korolkowii and C. alatavicus, both belonging to chemotype 1 (Table 2). However, they differ in contents of major flavonoids (Table 4). The systematic position of C. antalyensis (Series j) is interesting when considering anthocyanin and flavonoid patterns. The others are consistent with the grouping. Series l and m are only represented by one species each. In Series n three and two accessions of C. pulchellus and C. speciosus are included, respectively. In this series the chemical markers are in accordance with a common origin. The flavonoid pattern is uniform and the differences of anthocyanin contents are correlated with the colour, the observed variation may be caused by selective processes on subspecies level. The white flowered C. boryi (Series o) did not contain anthocyanins or at least the contents were below the detection limit (Table 2). However, it contains the same amounts of flavonoids as C. laevigatus, another species from same series (Table 4). The subgenus Crociris has only one species, C. banaticus, which differs from other species in several morphological traits and also in anthocyanin pattern (Table 2). The species contains group IV of the flavonoids (Table 4). In general, the chemical data support the morphological data, only rising some doubt about the placement of six taxa: C. medius from Series d should be further investigated because the results indicate that it may belong to Series c. C. asumaniae, C. hadriaticus and C. oreocreticus from Series f match both the anthocyanin and flavonoid patterns of Series c and may be closer to this series. C. mathewii in Series f shows similarities to Series e and g while C. siberi ssp. atticus in Series g differs from the other subspecies. 3.4. The anthocyanins and other flavonoids in species and cultivars of Crocus Series Biflori (h) The yellow-flowered C. chrysanthus has the same chemotype as both white and yellow-flowered C. danfordiae. Whether some of the different C. biflorus subspecies, which comprises five different chemotypes, should have rank as species, is difficult to say but many have previously been regarded as so. Nineteen cultivars in Series h seem to be C. chrysanthus × C. biflorus ‘Major’ hybrids, as they posses the same simple anthocyanins (A5 and A6). C. biflorus ssp. crewei only has A4 as a minor component, which indicates close relation to C. biflorus ssp. melantherus and C. chrysanthus. Other C. biflorus subspecies are characterised by a combination of many compounds, which is an important dissimilarity from the C. chrysanthus chemotype. Within the Series Biflori (h) most accessions fall in flavonoid group IV, both

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species, subspecies, and cultivars. However, C. biflorus ssp. crewei, C. biflorus ssp. adamii ’Serevan’ and C. biflorus ssp. taurii differ from the other subspecies (Table 5). The pigments indicate that the annulate species in Series n is a well defined series which is separated from the annulate species in Series h. In every other series than h, a relation between the chemical markers and the division of species was found. The diversity of chemotypes in Series h indicates that C. biflorus may include different species and a further division may be a suggestion. 3.5. The anthocyanins in hybrids The pigment patterns of our own documented artificial hybrids are shown in Tables 3 and 5 and their parental species are included in Tables 2 and 3. The parents of the yellow-flowered Crocus cultivars C. ‘Stellaris’ and C. ‘Golden Yellow’ are reported to be C. flavus and C. angustifolius, based on morphological traits and by genomic in situ hybridisation (Ørgaard et al., 1995b). The diploid hybrid C. ‘Stellaris’ (A1–A5), although A6 was not detected, has most of the parental anthocyanins: C. flavus (A5) and perhaps the other parent is C. angustifolius ’Minor’ (A1– A6 (trace)) rather than C. angustifolius (A3–A6 (low)) (Table 2). The flavonoid patterns of the parental species and the hybrid do not give any informations as to the origin. Both C. angustifolius and C. angustifolius ‘Minor’ belong to flavonoid group II, III and IV, while the hybrid C. ‘Stellaris’ has major flavonoids from group III and IV (Table 4). The triploid hybrid C. ‘Golden Yellow’ (A2–A7), seems to have the parental anthocyanins of C. flavus (A5) and C. angustifolius ‘Minor’ (A1–A5, A6 (trace)) rather than C. angustifolius (A3–A5, A6 (low)), but has an additional trace of A7 (Table 2). The flavonoid pattern supplemented the results since the hybrid belongs to group III, while the parental C. flavus belongs to I and IV and both C. angustifolius and C. angustifolius ‘Minor’ are categorised in group II, III and IV (Table 4). A conclusion regarding the hybrids is that their chemical composition is not always a sum of the two parental anthocyanin components i.e. they have the majority, but may lack one or two components. The ability to produce relatively high amounts of F11–F13, F21–F23, F25 (group I) appear to be recessive. However, it seems that anthocyanin chemotype 1, the ability to make relatively high amounts of A5 and A6, is dominant. The loss of anthocyanins in hybrids may be the explanation for the sometimes diverse anthocyanin patterns found in otherwise morphologically related species and subspecies, i.e. it may be the result of previous hybridisations in the evolutionary history.

4. Conclusions Three malonated anthocyanins and six flavonol glycosides all seem to be unique for Crocus. All these compounds were widely distributed within the genus, the fla-

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vonoids occurred in every taxon examined, and so can be used as distinguishing markers for this genus. The diversity of the distribution of the chemical structures within the genus made it possible to use them as chemotaxonomical markers, in particular the anthocyanins. Evaluation of anthocyanin chemotypes and flavonoid groups, each generally support the classification by Mathew (1982), and using the two types of compounds the results reinforce each other. For all series except Series h the chemical data were in most cases very similar for all subspecies or accessions within a species, and chemotypes within a series were more similar than between series. However, for six species or subspecies, the analyses suggest that they should be further investigated using other methods, to evaluate their relations to other series or species, respectively. Analyses of the C. chrysanthus–C.biflorus complex in Series h showed a wide range of anthocyanin chemotypes, which could support the earlier view that the C. biflorus subspecies actually comprise several different species. Methoxylated and malonated anthocyanins are widely distributed in the genus, both occurring in several series in both sections, and a few species contain all the nine anthocyanins found. This indicates that the ancestral Crocus may have contained all the anthocyanin chemotypes and flavonoid groupes found in the genus today.

Acknowledgements We thank P.W. van Eeden for the donation of additional plant material. The work was supported by the Danish Natural Science Research Council (Grant No. 9502849).

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