Am. J. Bot. Visit Plant Cell Online
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (21)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jenczewski, E.
Right arrow Articles by Ronfort, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jenczewski, E.
Right arrow Articles by Ronfort, J.
Agricola
Right arrow Articles by Jenczewski, E.
Right arrow Articles by Ronfort, J.
(American Journal of Botany. 1999;86:677-687.)
© 1999 Botanical Society of America, Inc.

Evidence for gene flow between wild and cultivated Medicago sativa (Leguminosae)based on allozyme markers andquantitative traits1

Eric Jenczewski2, Jean-Marie Prosperi and Joëlle Ronfort

Laboratoire de ressources génétiques et d'amélioration des luzernes méditerranéennes, Unité de Recherche deGénétique et Amélioration des Plantes, INRA Montpellier, Domaine de Melgueil, 34130 Mauguio, France

Received for publication December 4, 1997. Accepted for publication October 15, 1998.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Genetic differentiation between co-occurring crops and their wild relatives will be greatly modified by crop-to-weed gene flow and variation between human and natural selective pressures. The maintenance of original morphological features in most natural populations of Medicago sativa in Spain questions the relative extent of these antagonistic forces. In this paper, we measured and compared the pattern of population differentiation within and among the wild and cultivated gene pool with respect to both allozymes and quantitative traits. Patterns of diversity defined three kinds of natural populations. First, some populations were intermediate with respect to both allozymes and quantitative traits. This suggests that crop-to-weed gene flow may have created hybrid populations in some locations. Second, some populations were different from all the cultivated landraces with respect to both allozymes and quantitative traits. This probably results from variable gene flow in space and in time, due to demographic stochasticity in either natural or cultivated populations. Third, differentiation from cultivated landraces was only achieved for the quantitative traits but not for allozymes in two populations. This suggests that natural selection in some locations may oppose gene flow to establish cultivated traits into the natural introgressed populations.

Key Words: crop-to-weed gene flow • Leguminosae • Medicago sativa; • population differentiation • polyploidy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Spatial patterns of genetic diversity result from a balance between antagonistic forces (Levin, 1988 ). The homogenizing effects of gene flow oppose substantial genetic differentiation from being established or maintained by genetic drift and/or heterogeneous natural selection (Slatkin, 1987 ). The outcome depends on the relative strengths of selection, genetic drift, and gene flow. Indeed strong selective pressures can generate steep locus clines even for high levels of gene flow (Antonovics and Bradshaw, 1970 ). In contrast, intense gene flow may also impose less-adapted genes on locally well-adapted populations (see Barton and Whitlock, 1996 , for details).

The relationship between a crop and its wild or weedy relatives is controlled through a similar balance between introgression and divergence (De Wet and Harlan, 1975 ). Gene exchanges have promoted the morphological convergence between some wild and cultivated populations of rice (Langevin, Clay, and Grace, 1990 , and references therein), while gametophytic competition has been shown to be effective in maintaining the phenotypic integrity of both forms of pearl millet (Robert et al., 1991 ). Selection can become disruptive with domestication (De Wet and Harlan, 1975 ; Small, 1984 ) since most agronomic traits (dwarfing, nonshattering infrutescence, etc.) are thought to be detrimental or maladaptive in the wild (Small, 1984 ; Ellstrand and Hoffman, 1990 ). Hybrids between wild and cultivated plants are often poorly adapted for survival in the parental environments (Heiser, 1973 ) and usually occupy disturbed man-made habitats associated with cultivation (De Wet and Harlan, 1975 ). Domestication should thus promote the establishment of premating reproductive barriers and thus reproductive isolation when hybridization results in strongly reduced fitness (Dobzhansky, 1940 ; see also Howard, 1993 ). However, the occurrence of ubiquitous hybridizations (Cucurbita: Kirkpatrick and Wilson, 1988 ; Zea: Doebley, 1990 ; Oryza: Langevin, Clay, and Grace, 1990 ; Raphanus: Klinger, Arriola, and Ellstrand, 1992 ; Beta: Santoni and Bervillé, 1992 ; Boudry et al., 1993 ; Setaria: Till-Bottraud et al., 1992 ; Chenopodium: Wilson and Manhart, 1993 ; Helianthus: Arias and Rieseberg, 1994 ; Whitton et al., 1997 ; Sorghum: Arriola and Ellstrand, 1996 ) suggests that most reproductive barriers are imperfect and that striking morphological differences are often irrelevant to reproductive affinity (Wilson, 1990 ). Crop-to-weed gene flow is mostly pollen mediated (Ellstrand and Hoffman, 1990 ) and thus requires cross-compatibility and overlapping flowering periods within a crop-weed complex (Klinger, Arriola, and Ellstrand, 1992 ). Hybridizations are not necessarily restricted to narrow zones of close parapatry, and long-distance gene dispersal up to hundreds metres away from the cultivated source has been reported (Klinger, Arriola, and Ellstrand, 1992 ; Arias and Rieseberg, 1994 ; Conner and Dale, 1996 ), notably for Medicago sativa (Saint Amand, Skinner, and Peaden, 1996 ).

Wild populations of Medicago sativa L. from Spain occur along roadsides and fields and grow in parapatry with cultivated plants in many locations. They overlap in flowering periods, share the same pollinators (honey bees, bumble bees, megachiles) and appear to be cross-compatible as experimental hybridizations are successful (Prosperi et al., 1996 ). Both forms are autotetraploid, outcrossing, and partially self-incompatible. Typical wild and cultivated plants have, however, long been distinguished on the basis of conspicuous morphological traits (Casellas, 1962 ; Delgado Enguita, 1989 , and references therein). Although a wide range of variability has been observed (Jenczewski et al., 1998 ), most wild plants are distinguished by a crawling growth habit and rhizomes.

This pattern raises the question of the maintenance of morphological integrity in wild populations despite their parapatry with cultivated related forms. In this paper, we examine whether this pattern results from reduced crop-to-wild gene flow or from strong selection pressures exerted against cultivated traits in some wild populations. Knowledge of population structure for different kinds of characters is useful to identify the relative importance of gene flow, genetic drift, and selection. While the level of differentiation at neutral loci only depends on a balance between gene flow and genetic drift (Slatkin, 1987 ), the pattern of variation for quantitative traits may also reflect the effects of selection. Therefore, comparing neutral and quantitative variation can help to determine the relative importance of natural selection and migration in the process of differentiation (Spitze, 1993 , and references therein; Long and Singh, 1995 ; Podolsky and Holtsford, 1995 ; Bonnin, Prosperi, and Olivieri, 1996 ; Karhu et al., 1996 ; Yang, Yeh, and Yanchuk, 1996 ). In this study, we infer the amount of gene flow from the pattern of differentiation between wild and cultivated populations revealed with allozymes. We also analyze 13 quantitative traits in order to compare neutral and quantitative variation and thus evaluate the role of natural selection in the maintenance of morphological integrity in wild populations.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Population sampling and experimental design
In 1985 and 1986, we collected seeds from 103 natural populations of Medicago sativa all over Spain (Prosperi, Delgado Enguita, and Angevain, 1989 ). Each sampled population consisted of mature pods haphazardously collected from more than 50 plants. These pods were mixed and threshed in the laboratory and used for a preliminary evaluation. As discussed in Jenczewski et al. (1998) , we detected a great deal of variability for both morphological and agronomic traits among populations. For the present study, a subset of 15 natural populations was chosen from these 103 populations so as to represent this whole range of morphological variability. Figure 1 shows their location in Spain.



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 1. Location of the 15 natural populations and sites where the five studied landraces originated (shaded areas). Filled triangle = populations that have been both allozymatically and morphologically surveyed; inverted open triangle = natural populations that have only been morphologically analyzed.

 
Seeds from five Spanish landraces of M. sativa [Alcaroches (Alc), Ampurdan (Amp), Aragon (Arg), Mediterraneo (Med), and Tierra de campos (TdC)] were kindly provided by I. Delgado (SIA-DGA-Zaragoza, Spain) and used as cultivated references. The five landraces are considered as representatives of all the cultivated alfalfas in Spain (Delgado Enguita, 1996 ). Aragon is the most important one and is cultivated in irrigated lands of the Ebro valley, in the agrarian regions of Duero, Center, Andalusia, and Extremadura. The other landraces are cultivated in specific restricted areas (Fig. 1; Michaud, Lehman, and Rumbaugh, 1988 ; Delgado, 1989 ). We analyzed two independent sets of seeds (2 x 20 plants) for each landrace and did not find any difference between sets for allozymes frequency and quantitative traits. Therefore, the two sets were pooled for each landrace and analyzed together.

Original seeds collected from natural populations (no "increasing" step) and seeds representing the five cultivated populations were put in petri dishes for 2 d to germinate, then potted in a randomized ten-block design on 4 May 1995. Forty individuals per population (four individuals/block) were grown in 10-L plastic pots under greenhouse conditions until 20 April 1996 and afterwards on field conditions with continuous individual watering.

All 15 natural populations and five cultivated landraces were used for measuring quantitative traits. The allozyme survey was implemented on a subset of nine natural populations (Fig. 1) and three cultivated landraces (Aragon, Mediterraneo, and Tierra de campos). Population Malzeville, which belongs to Medicago falcata, a related species of the same complex, was electrophoretically surveyed in order to calibrate the amount of differentiation. This population originated from the north-east of France because we did not find any pure wild population of M. falcata during the collections in Spain.

Quantitative traits
Thirteen quantitative traits were scored on each individual. Morphological measurements were performed on the three main stems of each plant at the date of first flowering in 1996. They included the length and the diameter of each stem, the area of the fifth subapical leaf on each stem and the shape of its central leaflet. The shape factor was estimated as (4{pi} x area) ÷ (perimeter)2. The number of rhizomes and the number of stems were scored on 3 April 1996 from 1 = no rhizome to 5 = numerous rhizomes and from 1 = 5–6 stems to 8 = more than 150 stems, respectively. Autumn regrowth and winter growth were measured on the basis of the dry matter produced on 25 October 1996 and 9 March 1997, respectively. The ability to regrow after the first cut (performed on 18 April 1996) was scored as the total sum of day-degrees accumulated since 18 April (calculated on base temperature 0°C) that was necessary for a plant to flower again in 1996. Vegetative productivity was estimated as the total dry matter produced from 9 March to 16 July 1997 over all successive cuts. Flowering time in 1997 was expressed as the total sum of day-degrees accumulated since 9 March that was necessary for a plant to flower in 1997. Spring growth and regrowth efficiencies were measured by the dry matter produced at flowering in 1997 related to the total sum of day degrees accumulated since 9 March, and by the dry matter produced on 16 July 1997 related to the total sum of temperature since the last individual cut, respectively.

Electrophoresis techniques
Fresh pieces of actively growing young leaf tissue were ground in cold 0.1 mol/L Tris-HCl pH 7.2 extraction buffer. The supernatant was absorbed onto filter paper wicks, kept frozen at -80°C overnight, and electrophoresed the next morning. The filter paper wicks were loaded onto 13% starch gels. We used the following enzyme systems: 6-phosphogluconate dehydrogenase (6PGDH), esterase (EST), isocitrate dehydrogenase (IDH), leucine aminopeptidase (LAP), malate dehydrogenase (MDH), and menadione reductase (MRD). The following buffer systems were used to assay the indicated enzymes: TRIS-citrate pH 7.0 for 6-PGDH and IDH; lithium-borate pH 8.0 for LAP, EST, and MRD; histidine pH 5.7 for MDH. Banding intensity was used to identify unbalanced heterozygotes. Segregation patterns of polymorphic loci were verified by analyzing progenies issued from crosses and selfing.

Quantitative trait analysis
Statistical analyses were performed using the SAS/STAT software package (SAS, 1989 ). The length and the diameter of each stem, the area of the fifth subapical leaf on each stem, and the shape of its central leaflet were measured three times on the same individual. Since the estimated repeatability coefficients (Falconer, 1989 ) were > 0.6 for all measurements, the average value over the three replicates was used in the subsequent analyses. Principal Components Analysis (Proc PRINCOMP) was performed on the correlation matrix of traits in order to explore the overall variability. Hierarchical clustering (average method: Proc CLUSTER) was carried out on the coordinates of every plant on the principal components whose eigenvalue was higher then 1. Pseudo-t and pseudo-F values were used to determine the optimal number of clusters. Multivariate analysis of variance (Proc GLM) was used to test whether populations differed from one another and comparisons by pairs of populations were implemented with the contrast method. Given the large number of comparisons, a sequential Bonferroni test (Holm, 1979 ) was used to adjust the probability of first-type error to the number of comparisons (Rice, 1989 ). Univariate analysis of variance (Proc GLM) and Duncan multiple range tests were performed to detect differences among populations for each trait.

Allozyme analyses
Within-population genetic diversity was estimated as the mean number of alleles per locus (A), the effective number of alleles [Ae = 1 - /(1 - H)], the percentage of polymorphic loci (P), considering a locus as polymorphic when the most common allele had a frequency <=0.95, and Nei's index (1973) of mean gene diversity (H). Departure from Hardy-Weinberg expectations was checked for each polymorphic locus in each population by a Fisher's exact test performed between the expected and observed size of each genotypic class. Differentiation was measured with the parameter {rho} described by Ronfort et al. (1998) . In autotetraploid organisms, this parameter is analogous to the "correlation between truly outcrossed mates" described for diploid populations (Tachida and Yoshimaru, 1996 ). Interestingly, this parameter does not depend on the proportion of double reduction in autotetraploids (i.e., {alpha}: independent distribution of sister chromatids into gametes; see Bever and Felber, 1992 ) and can thus be computed over loci. As shown by Ronfort et al. (1998) , the expected value of FST for an autotetraploid species in an island model of population structure is FST = 1/(1 + 8Nem), where Ne is the inbreeding effective population size, which depends on the real demographic size, the selfing rate, and the migration rate. FST thus depends on s and {alpha}. Under the island model of population structure, the expected value of {rho} is 1/1 + 2Nm, i.e., independent from s and {alpha}. Estimations of these parameters were computed using the ANOVA framework developed by Weir and Cockerham (1984) extended for autotetraploid organisms (Ronfort et al., 1998 ). Differentiation significance was checked through Fischer's exact tests (Raymond and Rousset, 1995 ) expanded for autotetraploids (M. Raymond, Institut des Sciences de l’évolution, Montpellier, France). Differentiation between groups of populations was assessed through a hierarchical analysis (Weir, 1996 ). Confidence intervals for the among-group differentiation were built through permutations (Sokal and Rohlf, 1995 ).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Quantitative traits
The principal component analysis extracted four components whose eigenvalue was higher than 1. They accounted for 75% of the overall variability (Table 1). The first principal component (PC1) was associated with traits measuring morphology (stem length, stem diameter, leaf area, number of rhizomes, number of stems), autumn regrowth, winter growth, and the ability to regrow after the first cut (Table 1). PC2 combined traits accounting for vegetative productivity, spring growth and regrowth efficiencies. PC3 accounted for the shape of the central leaflet and flowering time in 1997. PC4 weighted the length and number of stems and the ability to regrow after the first cut.


View this table:
[in this window]
[in a new window]
  		Table 1. Principal component eigenvalues for each character.

 
Hierarchical cluster analysis clearly opposed wild (cluster 1) and cultivated (cluster 2) plants. These two clusters were mostly discriminated by PC1 (Fig. 2). The ratio of the between-cluster variance to the within-cluster variance was 4.15 for PC1 and <0.005 for the three other principal components. These values were deduced from the nearest centroid sorting method (Proc fastclus; SAS, 1989 ) performed with two prespecified clusters corresponding to the two main clusters assigned from the average method. According to the PC1 eigenvector, the typical wild plants were rhizomatous, displayed low winter growth, a poor productivity, and poor regrowth ability. They produced numerous shorter stems and smaller leaves as compared to the cultivated ones.



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 2. Plots of populations on the first two principal components axes. The different symbols discriminate the different groups of populations (see text): filled diamond = group 1; filled triangle = group 2; open circle = group 3.

 
Given this partition, we have considered that populations were typically wild (group 1) or cultivated (group 2) when >90% of their individuals belong to cluster 1 or cluster 2, respectively (Table 2). Populations E064, E097, and E147 did not fit these criteria because of their intermediate composition (Table 2) and were assigned to a third group (group 3). Multivariate and univariate analyses of variance were performed within these three groups and are detailed below.


View this table:
[in this window]
[in a new window]
  		Table 2. Proportion of wild (cluster 1, C1) and cultivated (cluster 2, C2) plants for each population.

 
Typical wild populations (group 1)
Multivariate analysis of variance showed that typical wild populations significantly differed from one another (Pillai's trace = 1.15; Pr > F = 0.0001). Univariate analysis of variance detected significant differences for most traits with the only exception of traits measuring winter growth, vegetative productivity, and regrowth efficiency.

Multivariate pairwise comparisons (contrast method) showed that populations E032, E066, E107, E109, E112, E129, and E132 did not differ from one another, as compared to the overall variability among the typical wild populations. Population E039 only differed from E129 and E066.

The remaining four populations were significantly different from most of the others. Population E073 displayed few longer stems, more elongated leaves, a greater autumn regrowth, a shorter flowering time in 1997, and a lower growth efficiency. Most of these features account for introgressions from M. falcata, which can be ascertained by the presence of variegated flowers and C-shaped pods. Population E143 had more slender stems and smaller leaves (Table 3). Population E118 was characterized by a faster spring growth with a lower growth efficiency and a lower autumn regrowth. Population E062 had more numerous rhizomes and a faster spring growth.


View this table:
[in this window]
[in a new window]
  		Table 3. Character means for each wild population (group 1), F value, and level of significance. Means followed by the same superscript letter are not significantly different at {alpha} = 0.05.

 
Cultivated landraces (group 2)
Multivariate analysis of variance showed that the cultivated landraces differed from one another (Pillai's trace = 1.06; Pr > F = 0.0001). But contrary to group 1, univariate analysis of variance only detected significant differences for half of the traits (Table 4).


View this table:
[in this window]
[in a new window]
  		Table 4. Character means for each cultivated ecotype, F value, and level of significance. Means followed by the same superscript letter are not significantly different at {alpha} = 0.05.

 
Multivariate pairwise comparisons showed that Aragon did not differ from Mediterraneo (Pr > F = 0.011) nor from Ampurdan (Pr > F = 0.179) and that Alcaroches did not differ from Tierra de campos (Pr > F = 0.0965). The strongest univariate differences among landraces were found for traits measuring autumn regrowth and winter growth (Table 4). Aragon only differed from Mediterraneo for autumn regrowth, and from Ampurdan for stem diameter. Tierra de Campos and Alcaroches had a lower winter growth and produced rhizomes (see Table 4 for details).

Populations with intermediate composition (group 3)
Multivariate pairwise comparisons showed that only populations E064 and E097 significantly differed from one another (Pillai's trace = 0.40; Pr > F = 0.0001). The results of the univariate analyses of variance strictly agreed with the relative population composition (Table 2). Population E064 contained 75% of plants from cluster 1 and displayed, on average, smaller leaves, more slender and numerous stems, more rhizomes, and a lower winter growth (Table 5). Population E097 contained 75% of plants from cluster 2 and showed the highest winter growth, while population E147 displayed intermediate values for most traits (Table 5).


View this table:
[in this window]
[in a new window]
  		Table 5. Character means for each intermediate population, F value and level of significance. Means followed by the same superscript letter are not significantly different at {alpha} = 0.05.

 
Hierarchical cluster analysis was performed on the first principal component scores within populations E064, E097, and E147 in order to verify whether they only contained a blend of wild and cultivated plants. As illustrated on Fig. 3, these populations not only contained a mosaic of individuals that could be roughly assigned to the wild or the cultivated gene pools, respectively, but they also displayed a continuous range of plants with intermediate features. Individual survey showed that these "intermediate" plants carried both wild (winter growth), cultivated (stem length), and intermediate characters (leaf area, shape of the central leaflet, autumn regrowth).



View larger version (27K):
[in this window]
[in a new window]
  		Fig. 3. Individual plots on the first two principal components axis for some populations. Population E062 and landrace Aragon are used as reference for the wild populations and cultivated landraces, respectively. The different symbols used for populations E032, E064, E097, and E147 refer to the different groups of individuals detected with a hierarchical cluster analysis.

 
Allozyme survey
Three enzyme systems (EST, MRD, and MDH) were excluded from our analyses because they led to complex banding patterns, which could not be interpreted unequivocally. The remaining systems resolved five loci (IDH, PGD1, PGD2, LAP1, and LAP2) that were polymorphic over all populations.

Within-population analysis
In most populations and for most loci, one allele was usually much more common than the others. The effective number of alleles per locus was therefore smaller than the average number of alleles (Table 6).


View this table:
[in this window]
[in a new window]
  		Table 6. Mean number of alleles per locus (A), mean effective number of alleles (Ae), proportion of polymorphic loci (P), and Nei's index for mean gene diversity (H) for each population.

 
The cultivated landraces displayed significantly less within-population genetic diversity (Ae, H: Table 6) than most natural populations (Wilcoxon rank test: P = 0.0126 for Ae and P = 0.0265 for H). Natural population E064 (group 3), however, displayed a level of genetic diversity similar to that of the cultivated landraces (Table 6).

Fisher's exact tests show that populations did not significantly depart from Hardy-Weinberg equilibrium. However, genotypic frequencies observed at the locus LAP1 did not fit the Hardy-Weinberg's expectations for six populations (E147, E143, E129, E066, E062, and E032). This result was probably due to the presence of null alleles that have already been reported at this locus in M. sativa (Birouk and Dattée, 1989 ) and in annual diploid species of Medicago (unpublished data).

Among-population analysis
We did not find any "diagnostic" alleles (i.e., common in some populations but absent from the others) to distinguish between M. falcata (Malzeville) and M. sativa, nor between the cultivated landraces and the natural populations. The genetic differentiation between all populations was statistically significant ({rho} = 0.1865 {Leftrightarrow} FST = 0.0865; P < 0.0001). The strongest differentiation was observed between Malzeville (M. falcata) and all the populations of M. sativa (Table 7). The value of {rho} estimated over all populations was approximately halved when Malzeville was removed from the analysis ({rho} = 0.1091 {Leftrightarrow} FST = 0.0490; P < 0.0001).


View this table:
[in this window]
[in a new window]
  		Table 7. Matrix of {rho} computed by pairs of populations. Significance was estimated by a Fischer's exact test.

 
The Spanish landraces were not significantly differentiated from one another ({rho} = 0.0140 {Leftrightarrow} FST = 0.0052; P = 0.3547). The natural populations (groups 1 and 3) were significantly differentiated from one another ({rho} = 0.0886 {Leftrightarrow} FST = 0.0409; P < 0.0001), but when considered as a whole they were not differentiated from the cultivated ones ({rho}among group = 0.0297 {Leftrightarrow} FST among group = 0.0135 ns). These results are due to: (1) the strong differentiation found between population E039 and all the other natural or cultivated populations, (2) the close proximity of populations E032 and E066 with the cultivated landrace Aragon, and (3) the lack of differentiation between populations E064 and E147 and all the cultivated landraces (Table 7). When considered as a group, populations E064 and E147 (group 3) were neither differentiated from the typical wild populations ({rho}among group = 0.0196 {Leftrightarrow} FST among group = 0.0092 ns) nor from the cultivated ones ({rho}among group = -0.0085 {Leftrightarrow} FST among group = -0.0035 ns). When populations E039, E064, and E147 were removed from the analysis, the remaining typical wild populations now appeared significantly differentiated from the cultivated ones ({rho}among group = 0.0879 {Leftrightarrow} FST. among group = 0.0191**).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Combining patterns of diversity based on allozymes and quantitative traits suggests three kinds of natural populations. First, populations E064 and E147 were not differentiated from all the cultivated landraces for allozymes and displayed intermediate quantitative features. Second, populations E032 and E066 were different from all the cultivated landraces with respect to quantitative traits but were not significantly differentiated from one of them, Aragon, for allozymes. Third, the remaining populations were different from all the cultivated landraces with respect to both allozymes and quantitative traits. These three kinds of populations suggest, respectively, that (1) gene flow has occurred from the cultivated landraces (notably Aragon) into several natural populations, (2) heterogeneous selection is likely to prevent the persistence of cultivated traits into natural populations, and (3) the amount of gene flow varies in time and/or in space.

Evidence for the occurrence of crop-to-weed gene flow
Populations E064 and E147 were neither differentiated from the typical wild populations nor from the cultivated landraces with respect to allozymes. This result strongly suggests that gene flow occurred from crop to wild populations. Though these patterns of differentiation could simply have resulted from a balanced blend of wild and cultivated individuals, several pieces of evidence attest that populations E064 and E147 were likely to be of hybrid origin.

First, populations E064 and E147 were not only intermediate for allozymes but also for quantitative traits (Tables 4–6GoGo). This trend was not due to linkage disequilibrium because there was no significant association between the quantitative traits and the allozymic loci (data not shown). Second, populations E064 and E147 contained a continuous range of plants with intermediate features (Fig. 3), which is generally recognized as a proof for hybridization and introgression (Jensen et al., 1993 ; Albert, d'Antonio, and Schierenbeck, 1997 ; see, however, Heiser, 1973 ; Rieseberg and Ellstrand, 1993 ). Third, genotypic frequencies in populations E064 and E147 were not different from those expected under random mating, which suggests that there was no positive assortative mating (i.e., homogamy) despite their heterogeneous composition (Fig. 3). Fourth, population E064 displayed a similar low level of genetic diversity as the cultivated landraces (Table 7). This reduced amount of diversity is exactly the trend we would expect when gene flow occurs from a genetically depauperate cultivated source into a variable sink natural population (Ellstrand and Elam, 1993 ).

As an alternative to hybridization, populations E064 and E147 could be viewed as the remnants of an ancestral form out of which the natural populations and the cultivated landraces have differentiated (e.g., Heiser, 1973 ; Small, 1984 ). This hypothesis is very unlikely because domestication of M. sativa began in the second millennium BC, in the Armenian highlands (Michaud, Lehman, and Rumbaugh, 1988 ) long before its first introduction into Spain in the first century BC, from Italy (Bolton, 1962 ). The present-day landraces are thus more likely to be derived from preexisting domesticates introduced into Spain through the successive waves of human settlement (Bolton, 1962 ; Michaud, Lehman, and Rumbaugh, 1988 ; Prosperi, Guy, and Balfourier, 1995 ), rather than from a wild native forms.

Finally, further support for the occurrence of gene flow is that Aragon was both the most widely and intensively cultivated landrace in Spain (Michaud, Lehman, and Rumbaugh, 1988 ) and the only one that was not significantly differentiated from four (out of nine) natural populations for allozymes (Table 7). This observation is notably true for populations E032 and E066.

The putative effects of natural selection
Populations E032 and E066 were not significantly differentiated from Aragon for allozymes but were different from all the cultivated landraces with respect to quantitative traits. They only contained wild-type individuals.

A large number of studies have now been published that detail the comparative divergence of phenotypic and allozymic characteristics (Spitze, 1993 , and references therein; Long and Singh, 1995 ; Podolsky and Holtsford, 1995 ; Bonnin, Prosperi, and Olivieri, 1996 ; Karhu et al., 1996 ; Yang, Yeh, and Yanchuk, 1996 ). Most of these have reported strongest differences with analyses of quantitative traits data than with electrophoretic ones and have suggested that natural selection acting upon the quantitative traits is responsible for the different patterns of differentiation revealed by neutral and quantitative traits. In our study, the contrasting patterns of variability observed in populations E032 and E066 may suggest that gene flow occurred from crop to wild populations and that strong divergent selection eliminated the cultivated traits from natural populations. It is worthwhile noting that the traits we measured in this study are likely to reflect divergent selective pressures between the wild and cultivated populations. Erected growth habit, productivity, growth, and regrowth efficiencies have been selected by man for hay production under irrigation. These traits are likely to be maladaptive in the wild because they are water consuming (Bolton, 1962 ; Melton, Moutray, and Bouton, 1988 ) and thus detrimental for survival in the dry areas where natural populations usually settle in.

Though this study does not provide direct estimate of the strength of natural selection, it does suggest that selective pressure may play a significant role in maintaining the morphological originality of populations E032 and E066. Controlled transplant experiments are now required to confirm this hypothesis.

The rate of crop-to-weed gene flow is variable
The remaining populations were significantly different from all the cultivated landraces for both allozymes and quantitative traits, although the pairwise amounts of allozymic differentiation often remained low (Table 7). This limited population structure is likely to account for the very slow genetic drift that may be expected for an autotetraploid allogamous organism like M. sativa (Haldane, 1930 ; Moody, Mueller, and Soltis, 1993 ).

The differences observed between natural and cultivated populations for both allozymes and quantitative traits suggest that these natural populations were isolated with regard to the cultivated landraces. The occurrence of gene flow between wild and cultivated populations of Medicago sativa thus varies in space and/or in time. This variation may be due to a combination of factors. Cultivated landraces usually suffer from a rapid turnover (every 3–4 yr) due to their rotation with other crops, while the natural populations occupy open manmade habitats (roadside, orchard) that are frequently disturbed. The episodes of parapatry between cultivated landraces and natural populations are therefore continuous neither in time nor in space so that crop-to-weed gene flow is locally and temporarily restricted. Also, cultivated landraces are mainly used for hay (Michaud, Lehman, and Rumbaugh, 1988 ) and usually harvested whenever the first flowers appear (10% flowering), because this schedule allows consistent forage yield and quality. Thus, the size of the "cultivated pollen pool" is often reduced and this should limit the extent of gene flow from crop to wild populations. Finally, natural populations are sometimes dense and continuous. The foraging pollinators are then likely to spend more time within these large populations, reducing proportionately the rate of interpopulation mating (Pyke, 1984 ). For a given distance between crop and weeds, the rate of pollen gene flow effectively decreases as the size of the recipient weed population increases (Klinger, Arriola, and Ellstrand, 1992 ).

Theoretical developments predict that variable migration rate or population size allow for the establishment and maintenance of stronger differentiation among populations (Whitlock, 1992 ; Barton and Whitlock, 1996 ). They could thus be relevant to the maintenance of the integrity of wild populations, in combination with selective pressure.

Implications for the release of transgenic plants
Our study suggests that crop-to-weed gene flow occurred between wild and cultivated populations in Spain and that neutral alleles were maintained over time. Hybridization is also likely with the numerous other cross-compatible widely distributed species associated with M. sativa. Small (1984) reported that hybrid swarms and backcross hybrids occur in nature between domesticated ssp. sativa and wild ssp. falcata at both ploidy levels (2n and 4n). This was shown to result in a wide variety of intermediate forms, including M. varia, which is abundant in many Mediterranean areas. Medicago sativa is the world's most important forage crop (Michaud, Lehman, and Rumbaugh, 1988 ) and has already been transformed with an herbicide resistance marker gene (D'Halluin, Botterman, and De Greef, 1990 ). Our study thus calls for a more extensive characterization of the amount of crop-to-weed gene flow among the Medicago species, as a prerequisite to the release of genetically modified alfalfa plants.


   FOOTNOTES
 
1 The authors thank I. Delgado Enguita for his help with the collection of plant material, S. Roussel and F. Meziane for laboratory assistance, O. Soudière, D. Tauzin, and M. Vabre for technical support, M. Angevain and G. Génier for their help and assistance in field surveys, M. Raymond for his help in extending the exact test procedure to an autotetraploid organism, T. Bataillon and I. Olivieri for advice and comments, J. Thompson for help in content and language on submitted version of the manuscript. This work was supported by a grant from the Bureau des Ressources Génétiques. E. J. is supported by an ASC fellowship from the Institut National de la Recherche Agronomique. Back

2 Author for correspondence (tel: 334 67 29 06 19, fax: 334 67 29 39 90, e-mail: jenczewski{at}ensam.inra.fr ). Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Albert, M. E., C. M. D'antonio, and K. A. Schierenbeck. 1997 Hybridization and introgression in Carpobrotus spp. (Aizoaceae) in California. I. Morphological evidence. American Journal of Botany 84: 896–904.[Abstract]

Antonovics, J., and A. D. Bradshaw. 1970 Evolution in closely adjacent populations. VIII. Clinal patterns at a mine boundary. Heredity 25: 349–362.[ISI]

Arias, D. M., and L. H. Rieseberg. 1994 Gene flow between cultivated and wild sunflowers. Theoretical and Applied Genetics 89: 655–660.[ISI]

Arriola, P. E., and N. C. Ellstrand. 1996 Crop-to-weed gene flow in the genus Sorghum (Poaceae): spontaneous interspecific hybridization between jonhstongrass, sorghum halepense, and crop sorghum, S. bicolor. American Journal of Botany 83: 1153–1160.

Barton, N. H., and M. C. Whitlock. 1996 The evolution of metapopulations. In I. A. Hanski and M. E. Gilpin [eds.], Metapopulation biology: ecology, genetics and evolution, chapt. 9, 183–210, Academic Press, New York, NY.

Bennett, J. H. 1968 Mixed self- and cross-fertilization in a tetrasomic species. Biometrics 24: 485–500.[CrossRef][ISI][Medline]

Bever, J. D., and F. Felber. 1992 The theoretical population genetics of autopolyploidy. Oxford Surveys in Evolutionary Biology 8: 185–218.

Birouk, A., and Y. Dattée. 1989 Organisation de la variabilité enzymatique des populations marocaines de luzerne (Medicago sativa L.): structures géniques et génotypiques. Genome 32: 120–128.[ISI]

Bolton, J. L. 1962 Alfalfa: botany, cultivation, and utilization. Leonard Hill Ltd., London.

Bonnin, I., J. M. Prosperi, and I. Olivieri. 1996 Genetic markers and quantitative genetic variation in Medicago truncatula (Leguminosae): a comparative analysis of population structure. Genetics 143: 1795–1805.[Abstract]

Boudry, P., M. Mörchen, P. Saumitou-laprade, P. Vernet, and H. van Dijk. 1993 The origin and evolution of weed beets: consequences for the breeding and release of herbicide-resistant transgenic sugar beets. Theoretical and Applied Genetics 87: 471–478.[ISI]

Casellas, J. 1962 El género Medicago L. en España. Collectanea Botanica VI(I-II): 183–291.

Conner, A. J., and P. J. Dale. 1996 Reconsideration of pollen dispersal data from field trials of transgenic potatoes. Theoretical and Applied Genetics 92: 505–508.[CrossRef][ISI]

de Wet, J. M. J., and J. R. Harlan. 1975 Weeds and domesticates: evolution in the man-made habitat. Economic Botany 29: 99–107.[ISI]

Delgado Enguita, I. 1986 The lucerne in Spain: characterization of the cultivated and spontaneous ecotypes. In G. Génier and J. M. Prosperi [eds.], The genus Medicago in the Mediterranean region: current status and prospects in research, Cahiers Options méditerranéennes, vol.18, 65. Centre International de Hautes Etudes Agronomiques Méditerranéennes, Zaragoza.

———. 1989 Estudio de la variabilidad de las Mielgas aragonesas en ares de precipitacion annual inferior a 600mm. Ph.D. dissertation, Universitad Politecnica de Madrid.

D'halluin, K., J. Botterman, and W. de Greef. 1990 Engineering of herbicide resistant alfalfa and evaluation under field conditions. Crop Science 30: 866–871.[Abstract/Free Full Text]

Dobzhansky, T. 1940 Speciation as a stage in evolutionary divergence. American Naturalist 74: 312–321.[CrossRef][ISI]

Doebley, J. 1990 Molecular evidence for gene flow among Zea Species. BioScience 40: 443–448.[CrossRef][ISI]

Ellstrand, N. C., and D. R. Elam. 1993 Population genetic consequences of small population size: implications for plant conservation. Annual Review of Ecology and Systematics 24: 217–242.[CrossRef][ISI]

———, and C. A. Hoffman. 1990 Hybridization as an avenue of escape for engineered genes. BioScience 40: 438–442.[CrossRef][ISI]

Falconer, D. S. 1989 Introduction to quantitative genetics. Longman, New York, NY.

Gliddon, C. 1994 The impact of hybrids between genetically modified crop plants and their related species: biological models and theoretical perspectives. Molecular Ecology 3: 41–44.

Haldane, J. B. S. 1930 Theoretical genetics of autopolyploids. Journal of Genetics 22: 359–372.[ISI]

Heiser, C. 1973 Introgression re-examined. Botanical Review 39: 347–366.

Holm, S. 1979 A simple sequencially rejective multiple test procedure. Scandinavian Journal of Statistics 6: 65–70.[ISI]

Howard, D. J. 1993 Reinforcement: origin, dynamics, and fate of an evolutionary hypothesis. In R. G. Harrison [ed.], Hybrid zones and the evolutionary process, chapter 3, 46–69. Oxford University Press, Oxford.

Jenczewski, E., M. Angevain, A. Charrier, G. Génier, J. Ronfort, and J. M. Prosperi. 1998 Contrasting patterns of genetic diversity in neutral markers and agro-morphological traits in wild and cultivated populations of Medicago sativa L. from Spain. Genetics Selection and Evolution. 30: 5103–5120.

Jensen, R. J., S. C. Hokanson, J. G. Isebrands, and J. F. Hancock. 1993 Morphometric variation in oaks of the apostle islands in Wisconsin: evidence of hybridization between Quercus rubra and Q. ellipsoidalis (Fagaceae). American Journal of Botany 80: 1358–1366.[CrossRef][ISI]

Karhu, A., P. Hurme, M. Karjalainen, P. Karvonen, K. Kärkkäinen, D. Neale, and O. Savalainen. 1996 Do molecular markers reflect patterns of differentiation in adaptative traits of conifers? Theorethical and Applied Genetics 93: 215–221.[CrossRef]

Kirkpatrick, K. J., and H. D. Wilson. 1988 Interspecific gene flow in Curcubita: C. texana vs. C. pepo. American Journal of Botany 75: 519–527.

Klinger, T., P. E. Arriola, and N. C. Ellstrand. 1992 Crop-Weed hybridization in Radish (Raphanus sativus): effects of distance and population size. American Journal of Botany 79: 1431–1435.[CrossRef][ISI]

Langevin, S. A., K. Clay, and J. B. Grace. 1990 The incidence and effects of hybridization between cultivated rice and its related weed red rice (Oryza sativa L.). Evolution 44: 1000–1008.[CrossRef][ISI]

Levin, D. A. 1988 Local differentiation and the breeding structure of plant populations. In L. D. Gottlieb and S. K. Jain [eds.], Plant evolutionary biology, 305–329. Chapman and Hall, New York, NY.

Long, A. D., and R. S. Singh. 1995 Molecules versus morphology: the detection of selection acting on morphological characters along a cline in Drosophila melanogaster. Heredity 74: 569–581.

Melton, B., J. B. Moutray, and J. H. Bouton. 1988 Geographic adaptation and cultivar selection. In A. A. Hanson, D. K. Barnes and R. R. Hill [eds.], Alfalfa and alfalfa improvement, chapter 20, 595–620. American Society of Agronomy, Crop Science Society of Agronomy, and Soil Science Society of Agronomy, Madison, WI.

Michaud, R., W. F. Lehman, and M. D. Rumbaugh. 1988 World distribution and historical development. In A. A. Hanson, D. K. Barnes and R. R. Hill [eds.], Alfalfa and alfalfa improvement, chapter 2, 25–91, American Society of Agronomy, Crop Science Society of Agronomy, and Soil Science Society of Agronomy, Madison, WI.

Moody, M. E., L. D. Mueller, and d. E. Soltis. 1993 Genetic variation and random drift in autotetraploid populations. Genetics 134: 649–657.[Abstract]

Nei, M. 1973 Analysis of gene diversity in subdivised populations. Proceeding of the Natural Academy of Sciences, USA 70: 3321–3323.

Podolsky, R. H., and T. P. Holtsford. 1995 Population structure of morphological traits in Clarkia dudleyana I. comparison of FST between allozymes and morphological traits. Genetics 140: 733–744.[Abstract]

Prosperi, J. M., I. Delgado Enguita, and M. Angevain. 1989 Prospection du genre Medicago en Espagne et au Portugal. Plant Genetic Resources Newsletter 78/79: 27–29.

———, M. Angevain, I. Bonnin, E. Chaulet, G. Génier, E. Jenczewski, I. Olivieri, and J. Ronfort. 1996 Genetic diversity, preservation and use of genetic resources of the Mediterranean legumes: alfalfa and medics. In G. Génier and J. M. Prosperi [eds.], The genus Medicago in the Mediterranean region: current status and prospects in research, Cahiers Options méditerranéennes, vol.18, 65. Centre International de Hautes Etudes Agronomiques Méditerranéennes, Zaragoza.

———, P. Guy, and F. Balfourier. 1995 Ressources génétiques des plantes fourragères et à gazon. Institut National de la Recherche Agronomique, Paris.

Pyke, G. H. 1984 Optimal foraging theory: a critical review. Annual Review of Systematics and Evolution 15: 523–575.

Raymond, M., and F. Rousset. 1995 An exact test for population differentiation. Evolution 49: 1280–1283.[CrossRef][ISI]

Rice, W. R. 1989 Analyzing tables of statistical tests. Evolution 43: 223–225.[CrossRef][ISI]

Rieseberg, L. H., and N. C. Ellstrand. 1993 What can molecular and morphological markers tell us about plant hybridization? Critical Reviews in Plant Sciences 12: 213–241.[CrossRef]

Robert, T., R. Lespinasse, J. Pernès, and A. Sarr. 1991 Gametophytic competition as influencing gene flow between wild and cultivated forms of pearl millet (Pennisetum typhoides). Genome 34 195–200.

Ronfort, J., E. Jenczewski, T. Bataillon, and F. Rousset. 1998 Analysis of population structure in autotetraploid species. Genetics. 150: 921–930.[Abstract/Free Full Text]

Saint Amand, P. C., D. Z. Skinner, and R. N. Peaden. 1996 Risk of alfalfa transgene dissemination and scale effects. In Report of the 35th North American Alfalfa Improvement Conference, NAAIC, Oklahoma City, OK.

Santoni, S., and A. Bervillé. 1992 Evidences for gene exchanges betwwen sugar beet (Beta vulgaris L.) and wild beets: consequence for transgenic sugar beets. Plant Molecular Biology 20: 575–577.[CrossRef][ISI][Medline]

SAS. 1989 SAS\STAT user's guide, version 6, 4th ed., SAS institute, Cary, NC.

Slatkin, M. 1987 Gene flow and the geographic structure of natural populations. Science 236: 787–792.[Abstract/Free Full Text]

Small, E. 1984 Hybridization in the domesticated weed-wild complex. In W. F. Grant [ed.], Plant biosystematics, 195–210. Academic Press, Toronto.

Sokal, R. R., and F. J. Rohlf. 1995 Biometry. W. H. Freeman, New York, NY.

Spitze, K. 1993 Population structure in Daphnia obtusa: quantitative genetic and allozymic variation. Genetics 135: 367–374.[Abstract]

Tachida, H., and H. Yoshimaru. 1996 Genetic diversity in partially selfing populations with the stepping-stone structure. Heredity 77: 469–475.[ISI]

Till-Bottraud, I., X. Reboud, P. Brabant, M. Lefranc, B. Rherissi, F. Vedel, and H. Darmency. 1992 Outcrossing and hybridization in wild and cultivated foxtail millets: consequences for the release of transgenic plants. Theoretical and Applied Genetics 83: 940–946.

Weir, B. S. 1996 Genetic data analysis II. Sinauer, Sunderland MA.

———, and C. C. Cockerham. 1984 Estimating F-statistics for the analysis of population structure. Evolution 38: 1358–1370.[CrossRef][ISI]

Whitlock, M. C. 1992 Temporal fluctuations in demographic parameters and the genetic variance among populations. Evolution 46: 608–615.[CrossRef][ISI]

Whitton, J., D. E. Wolf, D. M. Arias, A. A. Snow, and L. H. Rieseberg. 1997 The persistence of cultivar alleles in wild populations of sunflowers five generations after hybridization. Theoretical and Applied Genetics 95: 33–40.[CrossRef][ISI]

Wilson, H. D. 1990 Gene flow in squash species. BioScience 40: 449–455.[CrossRef][ISI]

———, and J. Manhart. 1993 Crop/weed gene flow: Chenopodium quinoa Willd. and C.berlandieri Moq. Theoretical and Applied Genetics 86: 642–648.[CrossRef][ISI]

Yang, R. C., F. C. Yeh, and A. D. Yanchuk. 1996 A comparison of isozyme and quantitative genetic variation in Pinus contorta ssp. latifolia by FST. Genetics 142: 1045–1052.[Abstract]




This article has been cited by other articles:


Home page
 Am. J. Bot.Home page
K. Birnbaum, R. DeSalle, C. M. Peters, and P. N. Benfey
Integrating gene flow, crop biology, and farm management in on-farm conservation of avocado (Persea americana, Lauraceae)
Am. J. Botany, November 1, 2003; 90(11): 1619 - 1627.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Bot.Home page
S. Montes-Hernandez and L. E. Eguiarte
Genetic structure and indirect estimates of gene flow in three taxa of Cucurbita (Cucurbitaceae) in western Mexico
Am. J. Botany, July 1, 2002; 89(7): 1156 - 1163.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (21)
Right arrow