Biochemical Systernatics and Ecology, Voi. 19, No. 7, pp. 587-593, 1991. 0305-1978/91 $3.00 + 0.00 Printed in Great Britain. © 1991 Pergamon Press plc.

Variation in the Flavonoid Aglycone Mixture Excreted on the Leaves of Black Alder (Alnus glutinosa), from 12 Different Geographical Origins

C. DANIERE,* J. F. GONNETt$ and A. MOIROUD* *Laboratoire d'Ecologie Microbienne et tLaboratoire de Biologie Micromoleculaire et Phytochimie,

Universit6 Claude Bernard-Lyon I, 43, Bid du 11 Novembre 1918, F-69622 Villeurbanne Cedex, France

Key Word Index--Alnus gludnosa; Betulaceae; flavonoid variation; provenance trial; geographic variation.

Abstract--Black alders from 12 origins, growing at La Cantle plantation (Sologne, France), have been surveyed for the composition of the flavonoid mixture excreted on their leaves. They originate from natural sites located in Poland, Germany and France representing a N.E./S.W. transect across the natural range of Alnus glutinosa. The qualitative and quantitative distribution of 18 flavonoid aglycones among the specimens was analysed by Principal Component Analysis (PCA). This calculation applied to data recorded in a first sampling composed of 10 individuals from each of 12 geographical origins. A second matrix was prepared using 90 individuals from three provenances only, selected according to their different biomass productivity. Specimens in both samplings display an important heterogeneity in their fiavonoid profiles, but no general relationship appears between the flavonoid content of specimens and their geographical origin. Likewise, there was no relationship between the expression of the flavonoid metabolism of Alnus trees and their ability in biomass production. However, using flavonoid patterns analysis remains helpful for individual characterization of Alnus glutinosa.

Introduction The genus A/nus includes about 30 species of deciduous trees and shrubs, mainly distributed in the northern hemisphere. Within this genus, A/nus g/utinosa (black alder) is recognized as one of the best species for sylviculture [1 ]. It has the potential for rapid biomass production and can be regenerated by stump sprout [2]. Moreover, like all other alders, it has the ability to symbiotically fix nitrogen, and consequently to grow on nitrogen-poor soil [3]. Because of its wide natural range, black alder should provide a large genetic reservoir for selection of most suitable specimens for maximum intensive culture and wood fibre production. Worldwide, several groups are working on improving A/nus g/ut/nosa performances [1, 4-7]. Presently, the most often used method in selecting "superior" specimens remains provenance trials [1, 5-8]. The selected specimens can then be included in seed orchards, and cloned for the produc- tion of genetically superior clones [1]. However, a commonly encountered problem in tree improvement is the characterization of specimens. Recently, we presented a new attempt to characterize A/nus g/ut/nosa individuals by analysis of the flavonoid aglycones excreted on their leaves [9, 10]. Individual flavonoid content was shown to remain stable over the course of a growing season and over successive years; it was also unaffected by the culture conditions and the process of vegetative multiplication [10]. This study describes variation of flavonoid aglycones among alder specimens of 12 different provenances, growing under the same climatic and biological conditions. The relationships of their chemical fingerprint with the geographical origin of specimens and their growth characteristics are examined.

Materials and Methods The detailed description of the planting site, establishment procedures and the experimental design of the

1:Author to whom correspondence should be addressed.

(Received 11 January 1991 )

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plantation has been reported by Teissier du Cros and Bouvarel [6]. The leaf material was harvested in July 1987, during the fifth growing year of specimens, and analysed the same day. Extraction and flavonoid analyses have been performed according to the method previously described [9]. The Principal Component Analysis (PCA) mathematical treatment of data was performed by STAT-ITCF (V.4.0), statistical software, running on an IBM-AT computer.

Results Within the context of the French programme of improvement of Alnus glut/nosa for short-term biomass production, a provenance trial test was established on the site "La Cant@e", located in Sologne (20 km S.W. of Orl@ans, France), by INRA* in March 1982.

The surveyed specimens, from 12 different provenances, were collected in the plantation (Table 1). They originated from the natural range of Alnus glutinosa across a N.E./S.W. transect represented by Poland, Germany and France. They are currently being tested for their biomass production and other biological characteristics. For a flavonoid survey, 177 plants were sampled among this wide provenance trial as follows: 10 plants of each of the 12 provenances (except for Diessen and Saint Julien, nine and eight plants available, respectively); 20 additional plants of three provenances: Czeszewo (no. 5, Poland), Gatinais (no. 6, France), Rott (no. 9, Germany), were selected according to their biomass productivity in the provenance-trial test. Gatinais is reported as the most productive, Rott being the least with the mean performance being represented by Czeszewo [6].

The flavonoid mixture excreted on the leaves of individuals was extracted and analysed according to a standard procedure [9]. The flavonoids detected in the samples are listed in Table 2; they are numbered according to the reference list used in our previous papers [9, 10]. Compounds 4 and 10 were not recorded in the present survey but a new component (2b) appeared in some profiles. In addition, compounds 19 and 20 were finally not considered because their chromatographic spots were not always readily detectable. The relative amount of flavonoids present in each individual profile was expressed as a percentage of the total amount (= 100%). The flavonoid data of all the surveyed specimens have been collected into an original 177 rows x 17 columns table.t This data matrix was divided into two sub-units for Principal Compo- nent Analysis (PCA): (i) the sampling of 10 individuals of each of the 12 geographical provenances; (ii) the 90 individuals of the three provenances (5, 6, 9) selected on the basis of their biomass productivity.

The results of the first PCA calculation are presented in Figs 1 and 2. The first ordi- nation accounts for 53.7% of the total inertia (F1 = 29.6% and F2 = 24.1%). The third

~INRA: Institut National de la Recherche Agronomique. tThis table is not presented here but is available on request from the Editorial Office of Biochemical

Systematics and Ecology.

TABLE 1. THE 12 PROVENANCES OF BLACK ALDERS CULTIVATED AT "LA CANTI~E"

Provenance Country District Altitude n

1 Fraimbois France Lorraine 250 10 2 Amance France Lorraine 350 10 3 Babki Poland Pormorze 80 10 4 Borowa Poland Pormorze 80 10 5 Czeszewo Poland Promorze 100 30 6 Gatinais France Centre 80 30 7 Meissner FRG Hessian Hill 350 10 8 Diessen FRG Bavaria 615 9 9 Rott FRG Bavaria 500 30

10 Seeshaupt FRG Bavaria 350 10 11 Prusse DRG Hessian Hill 350 10 12 Saint Julien France Landes 22 8

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TABLE 2. FLAVONOID AGLYCONES DETECTED IN THE SURVEYED INDIVIDUALS OF ALNUS GLUTINOSA

Number Compound

1 Unidentified flavonol 2a Unidentified 2b Unidentified 3 Unidentified 5 Quercetin-4'-methyl ether 6 Luteolin-4'-methyl ether 7 Quercetagetin-6',4"-dimethyt ether 8 Kaempferol-3-methyl ether 9 6-Hydroxy-luteolin-6,4'-dimethyl ether

11 Quercetagetin-3,6,4'-trimethyl ether 12 Kaempferol-4'-methyl ether 13 Apigenin-4'-methyl ether 14 Quercetagetin-6,7,4'-trimethyl ether 15 6-Hydroxy-kaempferol-6,4'-dimethyt ether 16 Unidentified 17 Scuttellarein-6,4'-dimethyt ether 18 6-Hydroxy-kaempferol-3,6,4'-trimethyl ether

axis represents only 9.1% of the variation. Samples are arranged along the F1 axis mainly according to their relative amount of 3,6,4'-trimethyl ether of quercetagetin (11) and aglycone 16 (negative part of F1). Along F2, individuals are distributed according to a metabolic balance, the ends of which correspond to the strongest production of 6,4'-dimethyl and 3,6,4'-trimethyl ethers of kaempferol (15 and 18, lower part of the axis) vs 4'-methyl ethers of quercetin and quercetagetin-6-methyl ether (5 and 7, upper part of F2). According to the above-described metabolic features, some samples display extreme profiles and, accordingly, are graphically located close to the end(s) of factorial axes. These individuals are shown by underlined bold numbers in Fig. 1 and are individuals 101, 108 (from Prusse, Eastern Germany, station no. 11) and 115 (from Saint Julien, France, station no. 12), at the negative part of FI. Samples from Borowa (32, from Poland, station no, 4) and from Diessen (91, from Germany, station no. 8), at the lower part of F2. Sample 3, from Fraimbois (France, station no. 1). As for the rest of the samples, they remain strongly mixed in a continuous variation structure in F1 × F2 (Fig. 1) and F1 x F3 (Fig. 2) graphical planes with no real relationships of flavonoid patterns with their geographical origin. For instance, specimens from Meissner (station no. 7, Germany), Seeshaupt (station no. 10, Germany) and Amance (station no. 2, France) are scattered all over the whole area of the factorial diagrams. However, samples from two stations, no. 1 (Fraimbois, France) and no. 11 (Prusse, Eastern Germany) appear less heterogeneous, being assembled in groups at the ends of factorial axis 1. In the second sampling including more individuals (30 instead of 10) for each provenance, the previous heterogeneity in specimens from Czeszewo (station no. 5), Gatinais (station no. 6) and Rott (station no. 9) is strongly confirmed. The samples of the three origins are intimately mixed together in both the factorial ordinations: F1 (= 38.7%) × F2 (= 18.2%) (see Fig. 3) and F1 × F3 (= 12.1%).

Discussion In this study of Alnus glutinosa with individuals mainly selected according to their geo- graphical origin, an important diversity in the constitution of individual flavonoid profiles is displayed again. The most important result is that no outstanding flavonoid features can be definitively attached with the natural origin of the specimens. It must be emphasized that specimens of remote origins often display nearly identical or closely related patterns: it is especially the case of samples with extreme profiles such as 108 from Eastern Germany (Prusse) and 115 from southwest of France (Saint Julien).

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FIG. 1, PCA OF DATA MATRIX: GRAPHICAL DISTRIBUTION OF THE 117 INDIVIDUALS FROM 12 PROVENANCES OF ALNUS GLUTINOSA IN THE 1 × 2 FACTORIAL ORDINATION, (F1 is fac tor ia l ax is 1; F2 is f a c t o r i a l a x i s 2).

With more individuals surveyed for each of three provenances of distant location, a similar conclusion applies since their profiles are extensively variable, with no relation- ship to their provenance. In addition, since the trees of these three provenances are reported to present striking differences in their biomass production, no relationships between the expression of the flavonoid metabolism with this biological parameter can be reported. The present results appear to be consistent with those obtained in surveying black alders from naturally growing populations in Rh6ne-Alpes district

FLAVONOID AGLYCONES IN ALNUS GLUTINOSA 591

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(France) [9] or even developed from three sets of seedlings, each one being issued from seeds collected on a single mother tree [10].

As suggested earlier [9, 10], this natural diversity originates in the strictly allo- gamous reproductive mode of Alnus glutinosa, the effects of which are enhanced by the dispersal mode of its pollen (wind) and seeds (wind and water) [11]. Consequently, a similar variability probably affects other biological features in alders, including their biomass production. For instance, Robinson and Mize [12] have recorded a strong variation in wood quality of black alders from 13 provenances: the variability in samples of each provenance had a comparable extent to the variation displayed by samples of different origins. Similarly, Bajuk et al. [13] report consistent results, con- cerning growing parameters of alders from six provenances. Alders of other species, namely Alnus crispa [14, 15] and Alnus incana ssp. rugosa [16] have been reported to show an important diversity in their allozymic systems. Thus, natural plantings of black alders exhibit an important diversity in most of the features of individuals, including growing parameters, allozymes and flavonoid content. Consequently, a major contribution of the present work, showing an important natural diversity in alders whether they are from single locations or from widely dispersed sites, is to emphasize that the current method of collecting seeds according to their provenance does not

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ensure the real expected homogeneity of the collected material and of its progeny, especially for selecting purposes. As for using the analysis of flavonoid patterns in the characterization of Alnus glutinosa individuals, this method remains helpful in the recognition of selected samples. Since the best specimens in selection are multipli- cated by asexual reproduction, this method may also be suitable for the control of the homogeneity of clonal progeny [10].

Acknowledgements--Luc Bouvarel and Eric Teissier du Cros (INRA, Ardon, France) are thanked for their help throughout the course of this work.

References 1. Hall, R. 8. and Miller, G. A. (1983) in Proceedings Third North Central Tree Improvement Conference, p. 233.

Held at Wooster, Ohio. 2. Hall, R. B., Nabb, H. S., Maynard, C. A. and Green, T. L. (1979) Bot. Gaz. 140, $120. 3. Tarrant, R. F. and Trappe, J. M. (1971) PI. Soil, Special Volume, 335. 4. Funk, D. T. (1973) in Ecology and Reclamation of Devastated Land (Hutink, R. J. and Davis, G, eds), p. 483.

London. 5. Dewald, L. E. and Steiner, K. C. (1986) Silvae Genetica 35, 205. 6. Teissier du Cros, E. and Bouvarel, L. (1987) in Biomass for Energy and Industry, p. 76. Elsevier Applied

Science. 7. Genys, J. B. (1988) Castanea 53, 71.

FLAVONOID AGLYCONES IN ALNUS GLUTINOSA 593

8. Maynard, C. A, and Hall, R. B. (1980) in Proc. 27th Northeast For. Tree Improvement Conf., p. 184. 9. Gonnet, J. F. and Dani~re, C. (1989) Biochem. Syst Ecol. 17, 239.

10. Dani6re, C., Gonnet, J. F. and Moiroud, A. (1991) Biochem. Syst. Ecol. 19, 577. 11. Hagman, M. (1970) Varparanta 1, 1. 12. Robison, T. L. and Mize, C. W. (1987) Wood Fiber ScL 19, 73. 13. Bajuk, L. A., Gordon, J. C. and Promnitz, L. C. (1978) Iowa State J. Res. 52, 341. 14. Bousquet, J., Chelliac, W, M. and Lalonde, M. (1987) Genome 29, 345. 15. Bousquet, J., Chelliac, W. M. and Lalonde, M. (1987) Physiol. Plantarum 70, 311. 16. Huenneke, L. F. (1985) Am. J. Bot. 72, 152.