Evolution and differentiation of populations of Lotus corniculatus s.1. (Fabaceae) from the southern French Alps (Massif du Ventoux and Montagne de Lure)

J. REYNAUD Laboratoire de botanique et biologie cellulaire, Institut des sciences pharmaceutiques ei biologiques,

Universite' Lyon I , 8 Avenue Rockefeller, 69373 Lyon CEDEX 08, France

M. JAY Laboratoire de biologie micron~ole'culaire et phytochimie, Universite' Lyon 1, 43 Boulevard du 11 Novembre 1918,

69622 Villeurbanne, France

AND

S. BLAISE Laboratoire de syste'n~atique et ecologie ve'ge'tales, Universite' Paris XI 91405 Orsay CEDEX, France

Received March 1, 1991

REYNAUD, J., JAY, M., and BLAISE, S. 1991. Evolution and differentiation of populations of Lotus corniculatus s.1. (Faba- ceae) from the southern French Alps (Massif du Ventoux and Montagne de Lure). Can. J. Bot. 69: 2286-2290.

A chemical analysis of the polyphenolic patterns of diploid and tetraploid populations of Lotus corniculatus s.1. (Fabaceae) growing in the southern French Alps showed that diploids from Mont Ventoux and from Montagne de Lure belong to the same chemotype. On the other hand, tetraploids showed two different chemotypes. This paper suggests the following hypoth- esis about the evolution of Lotus corniculatus in this area since the last glaciation: diploids could be derived from a relic diploid population scattered by glaciation and restricted within very narrow areas, whereas the tetraploids originating from the diiploids were more competitive and radiated into larger areas cleared from ice.

Key words: Lotus corniculatus, birdsfoot trefoil, Fabaceae, polyploidy, polyphenolic metabolism, evolution.

REYNAUD, J., JAY, M., et BLAISE, S. 1991. Evolution and differentiation of populations of Lotus corniculatus s.1. (Fabaceae) from the southern French Alps (Massif du Ventoux and Montagne de Lure). Can. J. Bot. 69 : 2286-2290.

Une analyse chimique des profils polyphCnoliques de populations diploides et tCtraplo'ides de Lotus corniculatus s.1. (Faba- ceae) rCcoltCes dans les Alpes du sud de la France, a montrC que les diplo'ides du Mont Ventoux et de la Montagne de Lure appartenaient i un m&me chimiotype. Par contre, les tCtraplo'ides appartiennent a deux chimiotypes diffkrents. Dans ce rapport, des hypothises sont envisagCes, concernant 1'Cvolution de Lotus corniculatus dans cette rCgion depuis les derniires glacia- tions : les diplo'ides pourraient &tre les descendants d'une population diplo'ide morcelCe par les glaciations et repoussCe dans des tenitoires trks riduits, tandis que les tCtraplo'ides provenant de ces diplo'ides, plus compCtitifs, se seraient rCpandus sur de larges surfaces libCrCes par les glaces.

Mots cle's : Lotus corniculatus, lotier cornicule, Fabaceae, polyplo'idie, mCtabolisme polyphCnolique, Cvolution.

Introduction

During the last glaciation (Riss and Wiirm), the Alps were overlaid by wide glaciers. Most plant species were forced southwards in marginal refugia or survived in nunataks (16). These climatic pertubations might have caused dislocation of the species' areas and favored the origin and expansion of new polyploids when the relict populations invaded the Alps again after the glaciation (4, 5, 6 , 16). In the southern French Alps, Mont Ventoux and Montagne de Lure may have been marginal refugia in particular for Lotus corniculatus s.1.; indeed, dip- loids and tetraploids of this species are present in this area as previously described by Favarger (6), Blaise and Cartier ( I ) , and Cartier and Blaise (2, 3).

In Mont Ventoux diploids are found from 1700 m, usually in cold and unstable habitats (north-facing screes). Tetra- ploids, when growing as isolated populations, preferably col- onize more moderate sites (south-facing stable meadows) but they can sometimes be found in mixed populations with dip- loids in unstable habitats.

In Montagne de Lure, diploids are found in the same eco- logical setting and at the same altitude as in Mont Ventoux (1). Tetraploids, as in Mont Ventoux, prefer more protected sites, i.e., one that gives the top of the mountain a mosaiclike disposition where tetraploids can grow in the shelter of Juni-

perus nana Willd., whereas diploids colonize the sparse mead- ows among these shrubs.

The purpose of this paper was to study, in this geographical context, the distribution and the degree of chemical differen- tiation between the two cytotypes of Lotus corniculatus. Our study is based on the polyphenolic profiles that reflect habitat differentiation and level of ploidy (7, 12, 15, 18, 19).

In this first step, compounds were not isolated or identified. Conclusions were drawn from comparisons of polyphenolic profiles (8, 1 1, 17).

Material and methods Plant material

Stems and leaves of 136 individuals at the flowering stage were collected from Mont Ventoux (five different sites, 79 samples) and from Montagne de Lure (three different sites, 57 samples) in July 1985 and 1986 (see Table 1). For each individual, one sample was collected for chemical study, and a second sample was collected for morphological and chromosome investigations.

Chrortlosome examination In a previous study, S. Blaise and D. Cartier (unpublished data)

showed that a clear-cut discrimination between diploids (2n = 2x = 12) and tetraploids (2n = 4x = 24) was possible as for other species (9, 14) with the help of six morphometric characters known to be

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TABLE 1. Number and location of the samples collected

No. of samples

Place Date Total Diploids Tetraploids

Site I Mont Ventoux top,

1910 m Mont Ventoux, top

1910 m Site Ia

Mont Ventoux, top, 1910 m, facing north

Site I1 Mont Ventoux, down to

Malauckne, 1800 m Site 111

Mont Ventoux, down to Malauckne, 1700 m

Mont Ventoux, down to Malauckne, 1700 m

Site IV Mont Ventoux, below Mont

Serein, 1400 m Site V

Montagne de Lure, top, 1800 m

Site VI Montagne de Lure, towards

Sisteron, 1700 m Site VII

Montagne de Lure, pas de la Graille. 1500 m

*Except Nos. 64, 71, 72, and 76.

significantly correlated with the degree of ploidy. The most signifi- cant were (i) length of the calyx (including the teeth); (ii) pilosity density of the stem; (iii) length of terminal leaflet of the leaf that axillates the first inflorescence; (iv) width of terminal leaflet of the leaf that axillates the first inflorescence; (v) maximum number of flowers per inflorescence; and (vi) length of the cauline hairs.

A first sorting was done in the field according to these easily avail- able morphometric characters, and a chromosome count was subse- quently performed on a voucher to check the accuracy of the method or in case of doubtful samples. In this case, chromosome counts were performed on the dividing root tip cells stained by the Feulgen reaction.

Each peak was treated as a character and a principal components analysis (PCA) was performed on the data from both diploid and tetraploid samples (136 individuals). A separate analysis was done only on tetraploids from both mountains (55 samples from Mont Ventoux, 34 from Montagne de Lure).

To facilitate the comparison of the different polyphenolic behav- iours, mean phenolic profiles were calculated and drawn (Fig 2). These profile averages are synthetic profiles obtained by calculating the mean value of each peak in a given group (diploids from Ventoux, diploids from Lure, etc.).

Results Diploid-tetraploid metabolic differentiation: phenolic markers

The HPLC profile obtained for each plant is a fingerprint (13) that reveals the activity of its polyphenolic pathways. Moreover, it is possible to verify that this fingerprint is stable enough to allow the recognition of a given genotype from one year to another, independently from the place where the plant grows, as previously reported for other species (7).

The PCA of the 136 HPLC profiles (Fig. 1) permitted clear separation of the two cytotypes, based on the first two axes that account for 68.9% of the total variation.

In our study, several chemical variables are responsible for the differentiation between diploids and tetraploids, as shown

Phytochemical study Samples were stored individually in 30 mL of 100% methanol after

collection. Polyphenolic compounds were extracted and analyzed using high performance liquid chromatography (HPLC). Samples were extracted with 70 mL of boiling methanol-water (7:3) for 18 h. The methanol in which samples had been stored since collection was added to this hydroalcoholic extract, and after evaporation under reduced pressure, the residue was dissolved in 200 mL of boiling water for 1 night and extracted twice with 80 mL of butanol. This last extract was evaporated to dryness and taken up in a small volume of methanol. This methanol extract was analyzed using HPLC (detec- tion at 340 nm).

by the average polyphenolic profiles obtained for both cyto- Mathematical treatment

The chromatographic profile was determined for each individual. types: chemotype D (defined by compounds H, L, P, and Q)

Within each profile, 23 peaks (or groups of peaks) were characterized for diploids and chemotype T (defined by compounds C, D,

by their retention time (here coded alphabetically A to W) and quan- H 7 L> and Or E~ J, L j and P, for tetraplOids (Fig. 2). tified by means of their area. Each individual is thus defined by the In the PCA, diploids appear as a dense cloud, whereas the relative amounts of each phenolic compound, the sum of which is tetraploid distribution is much looser and even discontinuous. equal to 100 (10, 12, 17). The same conclusion can be drawn from the morphometric

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CAN. J . BOT. VOL. 69, 1991

DIPLOIDS: C h e m o t y p e D

FIG. 1. Principal components analysis (axes 1 and 2) of the 136 samples from Ventoux and Lure mountains in the southern French Alps. Axes 1 and 2 are 68.9% of the total variation. A, diploids from Ventoux; A, diploids from Lure; 0, tetraploids from Ventoux; 0, tetraploids from Lure; 0, discriminant phenolic markers. D, TV, and TL are different chemotypes.

characters of both cytotypes (S. Blaise and D. Cartier, unpub- lished data).

Comparison of diploids from the Ventoux and Lure Mountains Profiles of all the individuals from both mountains are almost

identical and superposable to the synthetic profile average obtained for the group of diploids (Fig. 2).

This similarity is also confirmed by the dense cloud-like distribution of the PCA in which individuals from both local- ities are well intermingled (Fig. 1).

Comparison of tetraploids from both mountains The PCA performed on all diploid and tetraploid individuals

revealed a major scattering of tetraploids (Fig. 1); for Ventoux and Lure, a large cluster was clearly defined from the diploids, with some individuals (six) among the diploids. Moreover, for Ventoux, a smaller group (nine individuals) is close but not mixed with the diploids.

When the tetraploids from both mountains were analyzed separately (Fig. 3), the data revealed the existence of two dis- tinct polyphenolic profiles corresponding to the populations of both localities. This differential genetic regulation is demon- strated as well by the average profile of tetraploids from Ventoux in which five peaks (C, D, H, L, and P) are promi-, nent (chemotype TV) compared with that of tetraploids from Lure in which four peaks (E, J , L, and P) are prominent (chemotype TL, Fig. 2). Moreover, the polyphenolic profile averages of the tetraploids from each of the two mountains correspond in fact to a large range of profiles.

VENTOUX

LURE

VENTOUX

C h e m o t y p e TV

LURE

C h e m o t y p e T L

TETRAPLOIDS

FIG. 2. Mean phenolic profiles.

Discussion Chemical aspects of microevolution of Lotus

A comparison between the phenolic profiles of diploids and tetraploids emphasizes the following points: (i) Generally, tetraploids show a different chemical pattern from that of dip- loids; moreover, tetraploids from two neighbouring mountains mav have different chemical ~rofi les. as shown between Ven- toui and Lure. However a fkw exceptional cases have been noted. (ii) For Ventoux as well as for Montagne de Lure, some tetraploids have maintained a diploid chemical profile. (iii) In the case of Ventoux, a group of tetraploids have an interme- diate chemical profile between authentic diploids and tetra- ploids (control chromosome counts and study of morphometric characters have shown they were not triploids, as one might think). Therefore, tetraploidization clearly seems to be a very recent process, as shown by the presence of tetraploids among diploids, and introgression processes between the two cyto- types are probably still ongoing.

Concerning the diploids, their polyphenolic metabolism is very stable and homogeneous, since they all belong to a chemotype characterized by four predominant compounds or groups of compounds. These diploids may have arisen from a relict diploid population that grew in the whole area before glaciation and was scattered and restricted within very narrow habitats. The reaction to this constraint might have been evo- lution towards tetraploidy. However, since tetraploids were

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FIG. 3. Principal components analysis (axes 1 and 2) of the 89 tetraploid samples from Ventoux and Lure mountains in the southern French Alps. Axes 1 and 2 are 65.5% of the total variation. a, tetra- ploids from Ventoux; 0, tetraploids from Lure; 0, discriminant phe- nolic markers.

better competitors and colonizers (4, 2 1 ) , diploid populations remained divided and restricted in extreme environments where tetraploids could not become established, whereas tetraploids radiated into considerably larger areas, occupying a variety of new habitats (5, 14).

In the inner Alps (Swiss Alps, for instance), diploids of L. corniculatus are found only above 2000-2200 m on car- bonate substrate (22, 23). If we compare the diploids from the inner Alps with those of this area, we notice that the latter behave differently since they grow as colonies on carbonate substrate from 1700 m. Thus, one may wonder whether both diploid groups originated from the same relict diploid popu- lation, or if their differences reflect two separate origins.

Relationship between chemical aspect and habitat Diploid plants, either from Mont Ventoux or from Mon-

tagne de Lure, grow in severe climatic situations and unstable places. They are so strictly adapted to this kind of environment that their transplantation to another site is difficult. Most sam- ples died when transplanted in the experimental garden of the FacultC des sciences in Orsay, while tetraploids were able to survive. This particular behaviour of the diploids and the sim- ilarity of their phenolic patterns may be attributed to their pre- cise metabolic adjustment to particular environmental condi- tions common to the two mountains. These plants seem to behave as specialists as is commonly the case at the end of an adaptative radiation process (5, 20).

Tetraploids usually grow in less severe conditions but are able to establish in a variety of sites: meadows of the Pas de la Graille, clearings planted with trees on the top of Lure, road

sides, etc. (1, 3). The various levels of phenolic differentiation observed between as well as within the Ventoux and Lure pop- ulations could be the illustration of this large colonizing ability.

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