Laboratoire Malherbologie et Agronomie, INRA, Dijon Cedex, France
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Abstract |
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Introduction |
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The model plant Arabidopsis thaliana is being increasingly used in molecular evolutionary studies. The different genes studied so far in A. thaliana can be classified according to their function as follows: genes involved in the recognition of the plant pathogen or abiotic stimuli (Kawabe et al. 1997
; Caicedo, Schaal, and Kunkel 1999
; Kawabe and Miyashita 1999
; Stahl et al. 1999
; Kuittinen and Aguadé 2000
; Aguadé 2001
), floral homeotic genes (Purugganan and Suddith 1998
, 1999
), and genes that encode catalytic enzymes (Hansftingl et al. 1994
; Innan et al. 1996
; Miyashita, Kawabe, and Innan 1998
; Kawabe, Yamane, and Miyashita 2000
).
Surprisingly, genes involved in the control of flowering time have almost never been studied for natural variation at the DNA level. Flowering time has nevertheless been shown to be highly variable in A. thaliana, is clearly related to fitness, and would be a main determinant of adaptation to environmental variation (Pigliucci 1998
). Conversely, recent progress has been made in understanding the genetic determinism of flowering time in A. thaliana. A large number of genes have been described, among which the single-copy gene FRIGIDA (FRI) seems to be of particular importance. The FRI locus acts synergistically with the Flowering Locus C (FLC) to cause late flowering (Sheldon et al. 1999
). FLC is the key gene involved in the initiation of flowering; it is negatively regulated by vernalization but positively regulated by FRI (Sheldon et al. 1999
). Ecotypes having functional alleles at the FLC but nonfunctional alleles at the FRI will have a shorter life cycle compared with the ecotypes having functional alleles at both loci. Moreover, by sequencing four ecotypes, Johanson et al. (2000)
identified two different deletions in FRI that disrupt the open reading frame and demonstrated that some of the early-flowering ecotypes carry one of these loss-of-function mutations. The exact function of the FRI protein is still unknown, although it contains two coiled-coil domains, the importance of which remains to be identified.
In this study, sequence variation at the FRI gene was analyzed for 24 field strains sampled in western Europe (France and the United Kingdom) and one seedbank ecotype (Ler, Poland). There is recent evidence that the populations we sampled originated from a single postglacial colonization "wave" from a Pleistocene refugium located in the Iberian Peninsula (Sharbel, Bernhard, and Mitchell-Olds 2001
). Nevertheless, these populations display a range of variation for flowering time (under greenhouse conditions) similar to that observed in a collection of 249 worldwide ecotypes (unpublished data). It is likely that the flowering time has been subjected to diversifying selection to adapt to the various kinds of habitats occurring in western Europe. Because of their longer vegetative growth phase, late-flowering plants can accumulate and allocate more resources for seed production. Late flowering in the absence of vernalization also allows the seedlings that emerge in autumn to overwinter as rosette plants. In contrast, early flowering would be advantageous under climates with a short favorable season for growth or in disturbed habitats. Early flowering may also be advantageous under climates with a long growing season if it allows several reproductive cycles to be achieved during a year.
Our study shows that many replacements and indels have occurred during the evolution of the FRI gene in western European ecotypes and that some of these variations have led to a loss of function. Our objectives are, therefore, firstly, to compare the level and pattern of sequence variation in the FRI region with that of other previously studied genes, secondly, to understand which neutral and selective forces acted to create this pattern, and finally, to examine the relationship between the polymorphism in the FRI region and the natural variation for flowering time.
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Materials and Methods |
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DNA Sequencing
The total DNA was isolated from young leaves using a CTAB method (Doyle and Doyle 1987
). The FRI gene was amplified as two overlapping segments. The two pairs of primers used were Fri1 (5'-GAAGACTAAAAAGAGCACACCATCACCCC-3') and Fri1R (5'-CATTCCCTTGATACTTGATTCAAC-3') to amplify the 5'-end of the gene, and Fri2 (5'-CGAAATTGTTGCTTGTCAGAACCAAATG-3') and Fri2R (5'-ATGAAGAGAATCCAGATGACCAAGAGCC-3')to amplify the 3'-end of the gene. PCR products were purified using the Qiaquick PCR purification kit (Qiagen). To eliminate sequence errors caused by the Taq DNA polymerase, PCR products from five independent reactions were pooled and used as a template for sequencing. DNA sequencing was carried out by MWG Biotech A.G. (Ebersberg, Germany). All sequence polymorphisms were visually rechecked from chromatograms, with special attention to singleton polymorphisms.
Data Analysis
The published FRI sequence of the ecotype H51, a derivative of the ecotype "Stockholm," was included in this study (GenBank accession number AF228499; Johanson et al. 2000
). The analyzed region was located between nucleotide positions 126 and 2936 of the H51 ecotype and encompassed the entire coding region of the FRI gene. The A. lyrata FRI sequence corresponding to positions 616 to 2838 in the H51 sequence was provided by H. Kuittinen (University of Oulu, Finland). Sequences were aligned using the Multalin program (Corpet 1988
). A neighbor-joining tree was constructed with Mega version 2.0 (Kumar et al. 2001
), using the genetic distances estimated by the Jukes and Cantor (1969)
method. Sequence polymorphisms in A. thaliana were analyzed using the DnaSP program version 3.52 (Rozas J. and Rozas R. 1999
). Nucleotide variation was estimated as nucleotide diversity (
, Nei 1987
) and 4Nµ (
, Watterson 1975
). The Tajima (1989)
test that compares these two measures of nucleotide variation was used as a test for mutation-drift equilibrium. Observed values of Tajima's D statistic were compared with empirical distributions generated by coalescent simulations under a neutral infinite-site model and assuming a large constant population size, as implemented in DnaSP (Rozas and Rozas 1999
). The distribution of observed pairwise nucleotide differences, or mismatch distribution, was calculated and graphically compared with the expected mismatch distribution under constant population size or population growth with no recombination (Rogers and Harpending 1992
). The minimum number of intragenic recombination events was calculated using the four-gametes test (Hudson and Kaplan 1985
), and the parameter C = 4Nc was estimated using the method of Hudson (1987)
. Rates of synonymous and nonsynonymous substitution in the coding region of FRI were estimated using two different methods, the approximate method of Nei and Gojobori (1986)
with the Jukes and Cantor correction for multiple hits and the maximum likelihood method developed by Goldman and Yang (1994)
. This latter method accounts for transition-transversion bias and codon usage bias. Standard errors for the rates of synonymous and nonsynonymous substitution estimated using the method of Nei and Gojobori (1986)
were obtained by the bootstrap method implemented in Mega version 2.0 (Kumar et al. 2001
).
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Results |
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Genealogical Relationships Among Ecotypes
The neighbor-joining tree (fig. 2
) showed weak support for allelic dimorphism at the FRI, in contrast to several other genes previously studied in Arabidopsis (Kawabe, Yamane, and Miyashita 2000
). A small group of sequences formed a separated cluster with a bootstrap value of 77%, whereas the remaining sequences were not strongly structured into groups. There was no relationship between the tree structure and the geographical origin of the ecotypes. Pairs of geographically related ecotypes were always separate on the tree, whereas some geographically distant ecotypes clustered together. Ecotypes with an interrupted, nonfunctional FRI sequence were scattered among all clusters on the tree.
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Polymorphism and Divergence at the FRI Coding Region
A sliding-window analysis was conducted to better examine the distribution of silent (i.e., noncoding and synonymous) and nonsynonymous variations along the FRI gene region (fig. 4
). Silent divergence between A. thaliana and A. lyrata was higher in the intronic region than in the exons, suggesting that selective constraints reduced the level of variation in the coding regions of FRI. Peaks of silent diversity within A. thaliana were present in two regions of the gene: first, in the 5'-flanking region, second in a large region that comprised the two introns and exons 2 and 3. In contrast, there were very few to zero silent variations along the first exon. Nonsynonymous variation was present mainly in the first exon, where two high peaks of nonsynonymous diversity were observed. The first peak overlapped with the first coiled-coil domain of the FRI protein. Only small peaks of nonsynonymous variation were found in exons 2 and 3.
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The ratio of nonsynonymous to synonymous substitution at the coding region of FRI between A. thaliana and A. lyrata was 0.36 (table 3
). This ratio was higher than those previously found at other genes in A. thaliana. For CAL, AP3, PI (Purugganan and Suddith 1998
), CHI (Kuittinen and Aguadé 2000
), FAH1, and F3H (Aguadé 2001
), the ratio of replacement to synonymous divergence varied between 0.038 (for F3H) and 0.25 (for CHI). However, the ratio found for FRI was still significantly less than one (table 3
), indicating that the FRI protein is constrained against amino acid changes at the between-species level. The pattern was quite different, however, at the within-species level. For exons 2 and 3, the synonymous diversity was about 10-fold higher than the nonsynonymous diversity (table 3
), suggesting that purifying selection was also acting at the within-species level on this part of the gene. In contrast, the first exon in FRI showed a very high level of nonsynonymous to synonymous diversity (
a/
s = 5 over 954 bp, table 3
). For the whole gene, the ratio of nonsynonymous to synonymous diversity was 0.76, a value higher than those previously found at other genes in A. thaliana. For ChiB (Kawabe and Miyashita 1999
), PgiC (Kawabe, Yamane, and Miyashita 2000
), CHI (Kuittinen and Aguadé 2000
), FAH1, and F3H (Aguadé 2001
), the
a/
s ratio varied between 0.05 (for ChiB and F3H) and 0.38 (for CHI). The coding region of FRI had a G+C content of 45.8% for exon 1 and 43% for exons 2 and 3. The effective number of codons (ENC) used in a gene, as defined by Wright (1990)
, ranges from 20 when only one codon is used for each amino acid to 61 when all synonymous codons are equally used. For the FRI gene, ENC was 58.3 in the first exon and 51.8 in the second and third exons. No codon bias that could cause an elevated ratio of nonsynonymous to synonymous diversity was thus present.
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To examine the relationships between amino acid changes within FRI sequences and variation in flowering time, we mapped the flowering phenotype of each ecotype on a haplotype tree based on nonsynonymous polymorphisms only (fig. 5
). The ecotype H51 had the consensus amino acid sequence, which from a comparison with A. lyrata was also found to be the ancestral sequence, and was used as a reference. Two ecotypes with nonfunctional sequences, LAC and VOU, could not be attributed to a unique position on the tree because of either homoplasy or recombination. They were thus discarded from the tree construction. Figure 5 showed that interrupted FRI sequences descended from a variety of intact FRI proteins. Among the 13 ecotypes with an intact FRI sequence, nine different FRI proteins that differed from each other by up to four amino acid changes were observed. There was some variation in the degree of earliness between ecotypes having identical FRI amino acid sequences (e.g., for ecotypes ALL1, CLA, and PON). This variation could be the result of either environmental effects or the effects of genetic polymorphisms elsewhere (see Discussion). Because the functional structural requirements of the FRI protein are unknown, it was impossible to predict which amino acid changes would modify its activity. Instead, we classified the observed changes according to Grantham's (1974)
physiochemical distance: changes corresponding to a distance higher than 100 (the mean distance) were classified as nonconservative. Figure 5
showed that most branches of the gene tree that connected a late-flowering ecotype to an early-flowering ecotype was associated with either a loss-of-function mutation or a nonconservative amino acid change.
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Discussion |
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The significantly negative Tajima's D values and the bell-shaped mismatch distribution indicated the presence of an excess of low-frequency polymorphisms. Another noticeable result was the large number of haplotypes. Using simulations of a coalescent process for a large constant population size under the neutral infinite-site model with no recombination, we could show that the observed number of haplotypes (20) was significantly larger than expected under demographic equilibrium and neutrality (P = 0.001, expected mean number of haplotypes is 13.1). A rapid demographic expansion or a past selective sweep could both account for these results (Tajima 1989
; Depaulis and Veuille 1998
).
Heterogeneity of Nucleotide Variation Along the Sequence of the FRI Gene
Demographic factors, such as a recent expansion, affect all genes and all regions of a gene equally. In contrast, selection directly affects the genetic diversity at a target site and modifies the genetic diversity at linked sites via hitchhiking effects. Selection is thus expected to result in heterogeneous patterns of genetic diversity among different genes and across the sequence of a given gene. In FRI both the amount of nucleotide diversity at nonsynonymous sites that are potential targets of selection pressures and the amount of nucleotide diversity at silent sites varied greatly along the gene sequence (fig. 4 ). In the second and third exons of the FRI there was a significantly lower rate of nonsynonymous than synonymous variation, which indicated that variation in this part of the gene has been driven mainly by purifying selection. A bias toward low-frequency polymorphisms was also observed in exons 2 and 3, as in other regions of the gene. This could reflect a recent demographic expansion of the A. thaliana in western Europe, but a recent relaxation of purifying selection on the nonfunctional variants of the gene may also have contributed to this pattern. In contrast to exons 2 and 3, a large coding region corresponding to the first exon in FRI showed a reduced level of synonymous variation associated with an excess of nonsynonymous polymorphisms, including changes between amino acids with quite different physiochemical properties and stop codons. As there is evidence that FRI is a single-copy gene (Johanson et al. 2000
), it is unlikely that these polymorphisms are actually neutral.
Mechanisms of Maintenance of Excess Nonsynonymous Mutations
A high degree of intraspecific amino acid polymorphism has already been found in other genes in A. thaliana, such as floral homeotic genes (Purugganan and Suddith 1998
, 1999
) and ChiA (Kawabe et al. 1997
). On the basis of the highly selfing nature of A. thaliana and its distribution as small scattered populations, it was proposed that most nonsynonymous mutations would be only slightly deleterious and could, therefore, be maintained by genetic drift in small populations (Ohta 1992
; Whitlock 2000
). Another explanation for the excess nonsynonymous polymorphisms in the first exon in the FRI would lie in a recent relaxation of purifying selection on this gene region. In conjunction with a recent demographic expansion in western Europe, this might have led to a large number of amino acid replacements occurring at low frequency. Such a process has previously been invoked to explain the elevated
a/
s ratio in the melanocortin 1 receptor in human populations from Europe as compared with Africa (Harding et al. 2000
).
Under these two different explanations, however, an elevated but still lower than one a/
s ratio is expected. In the first exon in the FRI, the significantly higher than one
a/
s ratio, therefore, strongly suggests that the excess amino acid polymorphisms are mostly adaptive and have been maintained by positive selection.
Effect of Local Positive Selection in a Subdivided Population
Two kinds of selection pressures are known to increase the level of variation in a gene: local selection and balancing selection (Nordborg, Charlesworth, and Charlesworth 1996
; Charlesworth, Nordborg, and Charlesworth 1997
). According to Charlesworth, Nordborg, and Charlesworth (1997)
, local selection would be difficult to distinguish from balancing selection in the absence of within-deme data because both kinds of selection would result in an excess of intermediate frequency polymorphisms. Their conclusion was based on a model of local selection at a single locus in a population subdivided into two demes. However, more complex circumstances would lead to other patterns of genetic diversity. It should be considered that first, several different mutations in a gene can potentially change its functional properties and, conversely, a same phenotypic change may be achieved via different mutations in a given gene. Thus, a single selection pressure can potentially affect several sites in a gene. Second, highly selfing species such as A. thaliana show a high degree of population subdivision. Therefore, we may hypothesize that positive selection in a set of isolated populations may lead to the maintenance of a large number of low-frequency nonsynonymous changes, as observed for the FRI gene. The lack of linkage disequilibrium among the observed nonsynonymous polymorphisms in FRI (data not shown) indeed suggests that they have appeared independently. The fact that the loss of function has appeared from at least eight different mutations also supports this scenario.
A point that remains unclear, however, is why so few synonymous polymorphisms were observed at exon 1. Local selection is indeed known to increase differentiation not only at selected sites but also at linked neutral sites via hitchhiking effects (Charlesworth, Nordborg, and Charlesworth 1997
). A likely explanation for this would be that local positive selection was relatively recent and was preceded by episodes of purifying selection that swept out the nucleotide diversity in this region of the gene (purifying selection probably also acted on exons 2 and 3, as discussed previously). A recent episode of selection would also explain why no accumulation of further nonsynonymous mutations was observed in interrupted sequences, despite the fact that the gene sequence should no longer be under selective constraint once a loss-of-function mutation has arisen.
High Level of Recombination at the FRI and Other Genes in A. thaliana
As noticed previously by Kuittinen and Aguadé (2000)
, a striking feature of nucleotide sequence studies in A. thaliana is the considerable discrepancy found among different genes for the estimated recombination parameter 4Nc and its ratio to the estimated mutation parameter 4Nµ. This ratio can reach extremely large values, whereas A. thaliana is known to display a very low outcrossing rate (Abbott and Gomez 1989
). One explanation for this, as proposed by Kuittinen and Aguadé (2000)
, lies in the possible heterosis conferred by recombination between individuals carrying slightly deleterious mutations. In the light of our results, a possible role of local selection may also be suggested. First, local positive selection may increase the apparent number of recombinations simply by enhancing the overall level of nucleotide variation. Second, in inbreeding species, even rare recombination events are probably an efficient way to create adaptive variation. For example, a loss of function for the FRI gene, conferring earliness, can result from a mutation as well as from a recombination event involving any already nonfunctional FRI allele. Thus, local positive selection may have maintained more recombination events (effective recombination) than expected under neutrality.
Identifying the Targets of Selection for Flowering Time
FRI is a regulatory gene that has been shown to increase the expression level of FLC, a gene encoding a MADS-box protein that acts to inhibit flowering (Sheldon et al. 1999
; Michaels and Amasino 2000
). Two loss-of-function mutations in the FRI (the indels at site 263 and 1511) were previously shown to induce early flowering in natural ecotypes (Johanson et al. 2000
). Our study confirmed and extended these results to six other loss-of-function mutations. These mutations were clearly associated with a life cycle of reduced length.
The functional effects of the observed amino acid changes at the FRI and their consequences for flowering time are much more hypothetical. Some early-flowering ecotypes had an intact FRI open reading frame, which suggests the following three possibilities. First, some of the observed amino acid replacements in the FRI gene could modify or even suppress the function of the FRI protein. Indeed, some replacements seemed associated with a transition from a late-flowering to an early-flowering phenotype (fig. 5 ). Second, mutations in the promoter region of FRI, which were not investigated here, could also lead to the nonexpression of FRI. Third, early flowering may be caused by the activity of other flowering time genes. Because FRI interacts synergistically with FLC, a loss-of-function mutation in FLC results in earliness, whatever the FRI allele (Michaels and Amasino 2000
), and amino acid changes at the FLC may also affect flowering time. Moreover, the FLC is controlled by at least two other pathways, the vernalization pathway and the autonomous pathway, and sequence variation at the underlying genes may also affect flowering time. Clearly, the joint analysis of amino acid changes at several genes involved in flowering time is needed to better characterize the evolutionary importance of the amino acid changes identified in natural FRI alleles. Nevertheless, the previous analyses by Johanson et al. (2000)
and our results clearly demonstrate that FRI is a major target of natural selection for flowering time in A. thaliana.
Phylogenetical reconstruction and the comparison of the levels of diversity between complete and interrupted sequences suggest that the loss-of-function mutations are more recent than most of the amino acid changes observed in the FRI. Mutations resulting in stop codons are less frequent than the mutations causing amino acid changes. One can hypothesize that selection for earliness first induced amino acid changes until a stop codon or indel knocked out the gene.
A Putative Scenario for the Recent Evolution of Flowering Time in A. thaliana
In their study of 40 Arabidopsis ecotypes, Johanson et al. (2000)
found a latitudinal gradient of flowering time across Europe. The majority of late-flowering ecotypes were from northern latitudes, whereas most of the early ecotypes were from central and eastern Europe. Thus, early flowering may be generally advantageous under the latitudes from which the ecotypes studied in the present article originated. In contrast, climatic conditions that prevailed during glaciation in the refugia were probably close to the present conditions in northern Europe. One could, therefore, hypothesize that late flowering was advantageous during the last glacial period in Europe and that purifying selection was then acting to maintain the functionality of the FRI protein. According to this scenario, selection for greater precocity occurred after the postglacial recolonization of Europe, when new conditions were encountered.
The presence of late-flowering phenotypes among our studied ecotypes can be explained in different ways. First, they may reflect adaptation to locally varying environmental conditions. Second, if selection for earliness is only recent, populations with nonoptimal phenotypes may still be present. Third, as human-induced dispersal is known to be a major factor in the recent spread of Arabidopsis, some of our studied ecotypes may be long-distance migrants.
It also cannot be ruled out that some early-flowering ecotypes may predate the postglacial expansion. Related haplotypes may have ended up at different locations during the postglacial recolonization, producing the lack of geographical structure we observed. Whether phenotypic variation of flowering time in A. thaliana actually reflects the adaptation to present local conditions remains an open question that a better ecological characterization of Arabidopsis natural populations would help to answer.
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Acknowledgements |
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Footnotes |
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Keywords: nucleotide diversity
selection
flowering time
Arabidopsis thaliana
Address for correspondence and reprints: Valérie Le Corre, Laboratoire Malherbologie et Agronomie, INRA, BP 86510, 21065 Dijon Cedex, France. lecorre{at}dijon.inra.fr
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