Molecular Population Genetics of Floral Homeotic Loci: Departures From the Equilibrium-Neutral Model at the APETALA3 and PISTILLATA Genes of Arabidopsis thaliana

Corresponding author: Michael D. Purugganan, Department of Genetics, Box 7614, North Carolina State University, Raleigh, NC 27695., michael_purugganan{at}ncsu.edu (E-mail)

Communicating editor: A. G. CLARK

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Molecular variation in genes that regulate development provides insights into the evolutionary processes that shape the diversification of morphogenetic pathways. Intraspecific sequence variation at the APETALA3 and PISTILLATA floral homeotic genes of Arabidopsis thaliana was analyzed to infer the extent and nature of diversity at these regulatory loci. Comparison of AP3 and PI diversity with three previously studied genes revealed several features in the patterning of nucleotide polymorphisms common between Arabidopsis nuclear loci, including an excess of low-frequency nucleotide polymorphisms and significantly elevated levels of intraspecific replacement variation. This pattern suggests that A. thaliana has undergone recent, rapid population expansion and now exists in small, inbred subpopulations. The elevated intraspecific replacement levels may thus represent slightly deleterious polymorphisms that differentiate distinct ecotypes. The distribution of replacement and synonymous changes in AP3 and PI core and noncore functional domains also indicates differences in the patterns of molecular evolution between these interacting floral regulatory genes.

THE evolutionary genetic basis of morphological differences between species remains one of the central issues in evolutionary biology (GOULD 1977 ; RAFF 1996 ). It has become increasingly clear from developmental genetic studies that classes of regulatory genes control morphological differentiation, and recent approaches have led to suggestions that evolutionary diversification at these developmental loci may contribute to interspecies differences in structure (PALOPOLI and PATEL 1996 ; SHUBIN et al. 1997 ; PURUGGANAN 1998 ). Despite its central relevance to the study of morphological diversification, little is known about the molecular population genetics of developmental pathways and the genes that comprise them.

Levels of genetic diversity at regulatory loci may govern the rates of morphological divergence and limit the degree to which selection at these genes can shape evolutionary change (PALOPOLI and PATEL 1996 ; RICHTER et al. 1997 ). The fate of mutations that arise in regulatory genes is governed, not only by selective pressures at specific loci, but also by the species' life history and population structure. The interplay between population history, breeding system, and selection determines the nature of molecular variation and delineates the mechanisms that lead to evolutionary diversification. Molecular population genetic analysis provides a context for examining how historical, demographic, and genetic processes are interwoven to shape evolutionary variation in genes (AVISE 1994 ; AQUADRO 1997 ).

We are studying the molecular population genetics of the floral developmental pathway in Arabidopsis thaliana, probing the nature of variation in regulatory genes that control flower differentiation in plants. Our focus has been on the floral homeotic loci that are members of the plant MADS-box regulatory gene family of sequence-specific DNA-binding transcriptional activators (MA et al. 1991 ; YANOFSKY 1995 ; RIECHMANN and MEYEROWITZ 1997 ). Two of these homeotic genes, APETALA3 and PISTILLATA, are partially responsible for petal and stamen specification in developing Arabidopsis flowers, and mutations at these loci result in the transformation of petals to sepaloid organs and stamens to carpel-like structures (JACK et al. 1992 ; GOTO and MEYEROWITZ 1994 ). AP3 and PI are related by a gene duplication event that took place prior to the origin of angiosperms (KRAMER et al. 1998 ), and their protein products appear to interact directly to control organ formation (MCGONIGLE et al. 1996 ; RIECHMANN et al. 1996 ).

Recent molecular studies have delimited the domains within both APETALA3 and PISTILLATA that are essential for the regulatory functions of these proteins (RIECHMANN and MEYEROWITZ 1997 ). Deletion and domain-swapping analyses have defined the core region of these proteins as consisting of the MADS-box, the I-region, and approximately the first 16 amino acids of the K-region (KRIZEK and MEYEROWITZ 1996A , KRIZEK and MEYEROWITZ 1996B ; RIECHMANN et al. 1996 ; see Figure 1). This core sequence is necessary for DNA-binding and dimerization activity of both AP3 and PI proteins and also appears to provide functional specificity. The other half of the protein, the noncore region, includes the rest of the K-box and the C-terminal sequence and may be involved in strengthening specific dimerization activities and in direct contact with the transcriptional machinery (RIECHMANN and MEYEROWITZ 1997 ).

View larger version (7K): In this window In a new window Download PPT slide   Figure 1. Schematic of structural and functional domains in AP3 and PI proteins.

Mutant analysis indicates that both AP3 and PI perform overlapping, nonredundant functions in stamen and petal development, and the comparable developmental roles played by these regulatory loci in floral morphogenesis suggest they may both evolve with similar dynamics. In this article, we determine the levels and distribution of nucleotide polymorphisms in AP3 and PI among different ecotypes of A. thaliana. Our results, coupled with those from other unlinked nuclear loci, provide insights into the history and population structure of this wild weed and allow us to explore the evolution of these two floral homeotic genes.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation and sequencing of alleles:
The A. thaliana ecotypes were obtained from single-seed-propagated material provided by the Arabidopsis Biological Resource Center (see Table 1). The Kent, Bretagne, Lisse, and Corsacalla seed stocks were from the population collection of P. H. Williams maintained at the Arabidopsis Biological Resource Center. A. lyrata seed was provided by C. H. Langley.

Miniprep DNA was isolated from young leaves as previously described (AUSUBEL 1992 ). PCR was performed with 40 cycles of 1 min at 95°, 1 min at 52°, and 3 min at 72° followed by 15 min at 72°. The error-correcting recombinant Tth polymerase XL formulation (Perkin Elmer, Norwalk, CT) was used to minimize nucleotide misincorporation. The error rate for this polymerase formulation, on the basis of multiple amplification and resequencing of known genes, is less than 1 in 7000 bp (M. D. PURUGGANAN and J. I. SUDDITH, unpublished results). We estimate that the nonsampling variance of nucleotide diversity due to PCR misincorporation, Var_PCR(), is negligible [Var_PCR()/Var() ~ 0.14] and does not significantly affect the frequency distribution of polymorphisms. The AP3-specific primers AP3F (for exon 1 forward), 5'-GAATATGGCGAGAGGGAAGATCC-3', and AP3R (for exon 7 reverse), 5'-GCCTTTAATTATTCAAGAAGATGG-3', and the PI-specific primers PI-1F (for exon 1 forward), 5'-GAGAAAGATGGGTAGAGGAAG-3', and PI-1R (for exon 6 reverse), ATCTCGATGATCAATCGATGACC-3', were used in PCR reactions to amplify alleles from A. thaliana and A. lyrata. The isolation of A. lyrata sequences will be reported elsewhere (A. L. LAWTON RAUH, E. S. BUCKLER and M. D. PURUGGANAN, unpublished results). Amplified DNA was cloned into pCR2.1 using the TA cloning kit (Invitrogen, San Diego). DNA sequencing for both genes was conducted with the ABI377 automated sequencer using a series of nine nested internal sense and antisense primers. All sequence polymorphisms were visually rechecked from chromatograms, with special attention to low-frequency polymorphisms (HAMBLIN and AQUADRO 1997 ). The DNA sequences are available from GenBank (accession numbers AF115798 to AF115830).

Data analysis:
Sequences used in this study were visually aligned. Phylogenetic analyses were conducted using PAUP 3.1 (maximum parsimony; SWOFFORD 1992 ). The heuristic search algorithm was utilized using the tree bisection-reconnection procedure, with the A. lyrata orthologues as the outgroup. Node support is assessed with 500 bootstrap replicates of the data. The polymorphism data was analyzed using the SITES (HEY and WAKELEY 1997 ) and DNASP (ROZAS and ROZAS 1997 ) programs. Levels of nucleotide diversity were estimated as mean pairwise differences () and number of segregating sites () (NEI 1987 ). Association between polymorphic nucleotide sites was tested using Fisher's exact test, corrected for multiple tests using the Bonferroni procedure (SOKAL and ROHLF 1981 ). Identification of possible recombinants utilized the four-gamete test (HUDSON and KAPLAN 1985 ). The Tajima (TAJIMA 1989 ) and Fu and Li (FU and LI 1993 ) tests for distribution of nucleotide polymorphisms were conducted without specifying an outgroup. Contingency tests for independence of mutational categories, referred to as the McDonald-Kreitman test (MCDONALD and KREITMAN 1991 ), were conducted using Fisher's exact test to evaluate significance. The coding region variation was also partitioned into core and noncore functional domains (RIECHMANN et al. 1996 ) for separate contingency analyses (TEMPLETON 1996 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Nucleotide variation at the Arabidopsis APETALA3 and PISTILLATA genes:
A total of 19 APETALA3 and 16 PISTILLATA alleles was isolated from a collection of 21 distinct, mostly European, A. thaliana ecotypes. Around 1.68 kb was sequenced for each APETALA3 allele, spanning exons one to seven and including 4 bp of the 5'-untranslated region (UTR) and 10 bp of the 3' flanking region of the gene. This sequence encompasses the entire AP3 coding region. Approximately 2.05 kb of sequence was obtained from PISTILLATA alleles. The PI sequences include the entire coding region (from exons 1 to 6 and intervening introns), 7 bp of the 5'-UTR, and 13 bp of the 3' flanking region of the gene. The APETALA3 and PISTILLATA genes encode proteins of 232 and 208 amino acids in length, respectively.

Both the APETALA3 and PISTILLATA genes in A. thaliana display considerable amounts of nucleotide variation (see Figure 2 and Figure 3). For AP3, a total of 78 nucleotide polymorphisms are present in the sampled alleles. These include 20 replacement polymorphisms that result in amino acid variation between alleles, while only 8 polymorphisms within the coding region are synonymous. There are 7 conservative and 13 radical replacement polymorphisms at AP3. The only insertion/deletion variations are associated with a (TG)_n microsatellite repeat in intron 5 of the gene, which differs in repeat length from 8 to 13 between alleles.

View larger version (36K): In this window In a new window Download PPT slide   Figure 2. Sequence of AP3 alleles from different Arabidopsis ecotypes. All allele sequences are compared to the reference allele from the Columbia ecotype. The position of the polymorphic sites and their locations in introns and exons are indicated at the top. The different amino acids encoded by replacement polymorphisms are shown below, with the most predominant amino acid shown first. The core and noncore regions are indicated.

View larger version (35K): In this window In a new window Download PPT slide   Figure 3. Sequence of PI alleles from different Arabidopsis ecotypes. All allele sequences are compared to the reference allele from the Landsberg erecta ecotype. The positions of the polymorphic sites and their locations in introns and exons are indicated at the top. The different amino acids encoded by replacement polymorphisms are shown below, with the most predominant amino acid shown first. The core and noncore regions are indicated.

The PI alleles reveal a total of 67 nucleotide changes, of which 16, 12 replacement and 4 synonymous, polymorphisms are found within the coding region. There are 5 conservative and 7 radical replacement polymorphisms in this gene. Five distinct insertion/deletion polymorphisms were observed in intron sequences, including two 1-bp indels. The Lisse-1 allele contains a unique 22-bp insertion in the first intron. The other two indels are associated with an interrupted microsatellite (TG)₃TCAG(TG)_n, where n = 6 to 8.

The estimates of sequence diversity at these two regulatory loci are comparable, although PI shows slightly less variation. The overall estimates of species-wide nucleotide diversity, , for AP3 and PI are 0.0064 ± 0.0008 and 0.0053 ± 0.0004, respectively. The estimate of for AP3 is 0.01319, with an upper bound of 0.0179 and a lower bound of 0.01169 at 95% confidence. For PI, the estimate for is 0.010 (upper bound is 0.0137, and lower bound is 0.00878).

The four-gamete test also indicates that at least one intragenic recombination has occurred in both AP3 and PI. In AP3, the Basel-1 allele may have originated from recombination between positions 1337 and 1372 in intron 6. The pattern of variation at the PI Lisse-1 allele is also consistent with recombination between positions 673 and 1900 (see Figure 2 and Figure 3).

Significant excess of low-frequency polymorphisms:
The distribution of polymorphic sites in both AP3 and PI is significantly skewed toward rare alleles. In the AP3 gene, 69 of the 78 nucleotide polymorphisms are found only once in the sample (singletons). The PI gene also shows a preponderance of low-frequency alleles—58 of 67 nucleotide polymorphisms at this locus are also singletons.

The skewness in the frequency distribution of polymorphisms is significant in both the TAJIMA 1989 and FU and LI 1993 tests. The Tajima test statistic D is -2.1507 for AP3 (P < 0.05); the negative value of the D statistic indicates that sampled alleles have an excess of rare alleles over that expected in an equilibrium population. The Fu and Li test statistic D* is also significantly negative for this gene (D* = -3.3728, P < 0.02). The same pattern is observed for the PI locus. For this gene, both the Tajima (D = -2.0183, P < 0.05) and Fu and Li tests (D* = -2.8755, P < 0.02) reveal that the distribution of polymorphisms for this regulatory gene is also significantly biased toward rare alleles.

Intraspecific gene genealogies of the AP3 and PI loci:
The genealogies of the naturally occurring alleles for APETALA3 and PISTILLATA are shown in Figure 4. Two classes of APETALA3 alleles are present in A. thaliana, suggesting that this gene exhibits allelic dimorphism in this species (see Figure 2 and Figure 4). The class A alleles, which are found in 12 of 19 sampled ecotypes, form a monophyletic group with 96% bootstrap support in the maximum parsimony tree. The rest of the alleles (referred to as the AP3 class B alleles) form an unresolved basal group in the phylogeny. The two allele classes are distinguished by four closely linked nucleotide polymorphisms in intron 4 (T to A at position 767, A to G at position 768, A to T at position 770, and T to G at position 779; see Figure 2). The mean pairwise differences () within the A and B allele classes are 0.00616 and 0.00448 differences/bp, respectively. The average pairwise difference between the two allelic classes is 0.00797 differences/bp.

View larger version (28K): In this window In a new window Download PPT slide   Figure 4. Gene genealogies of AP3 and PI alleles. All nodes with < 50% bootstrap support are collapsed; the other bootstrap values are indicated next to relevant nodes. Class A and B alleles for AP3 are indicated.

There is only weak support for allelic dimorphism at PI. Instead, PI alleles appear to be structured into four allele groups, which are differentiated from each other by only one to two shared nucleotide polymorphisms (see Figure 4). Both the AP3 and PI gene genealogies do not reveal any clear relationship of alleles with locality, and the differentiation into two allelic classes is not strongly correlated with geography. Moreover, the genealogies of the AP3 and PI alleles are not concordant with one another.

Nonrandom associations between polymorphic sites:
The four polymorphisms in APETALA3 that differentiate the A and B allele classes are in complete linkage disequilibrium with each other. Four segregating sites in a 13-bp region of intron 4 (positions 767 to 779) exhibit significant levels of nonrandom association with each other (P < 0.001, corrected for multiple tests; see Figure 5). In the PI locus, the strongest nonrandom association is between site 798 in intron 1 and position 1401 in intron 2 (P < 0.001, corrected for multiple tests). The polymorphisms at these two sites are also in complete linkage disequilibrium with each other. Because the products of these two loci physically interact during floral development, we also tested whether there is linkage disequilibrium among sites between the two regulatory genes. Analyses of the joint sequence of AP3 and PI, however, do not reveal any significant intergenic association of polymorphic sites.

View larger version (21K): In this window In a new window Download PPT slide   Figure 5. Linkage disequilibrium between polymorphic sites in AP3 and PI. The matrix indicates the pairs of sites that are nonrandomly associated with each other. The association between sites marked by the black squares is highly significant (P < 0.001). The dark gray and light gray squares indicate associations that are significant to the 0.001 < P < 0.01 and 0.01 < P < 0.05 levels, respectively.

Excess intraspecific replacement polymorphisms at AP3 and PI:
The protein-coding regions of the AP3 and PI genes are not evolving according to the predictions of the equilibrium neutral hypothesis (see Table 2). The AP3 gene appears to contain an excess of intraspecific replacement polymorphisms, and the McDonald-Kreitman test rejects the prediction of the equilibrium neutral theory that intraspecific polymorphisms and interspecific divergences are correlated. The AP3 gene has 20 replacement and only 8 synonymous polymorphisms, and comparison of A. thaliana and A. lyrata genes reveals only 6 replacement (4 conservative and 2 radical) and 14 synonymous differences. The excess of intraspecific replacement polymorphisms is significant for this locus (P = 0.005).

The PI gene also exhibits a significant excess of within-species replacement polymorphisms. There are 12 replacement and 4 synonymous polymorphisms at the PI locus of A. thaliana, which differs from the A. lyrata orthologue by 11 replacement (6 conservative and 5 radical) and 16 synonymous differences. The McDonald-Kreitman test also rejects the predictions of the neutral hypothesis for the PI gene (P = 0.03).

Distribution of sequence variation between functional domains of AP3 and PI:
The partitioning of nucleotide changes between core and noncore functional domains within the two genes (see Figure 1) can be examined by studying the number of within-species polymorphisms and between-species differences (see Table 2). The AP3 gene has an excess of within-species replacement polymorphisms in both the core (5 replacement to 4 synonymous) and noncore (15 replacement to 4 synonymous) regions, with a threefold increase in replacement variation in the latter domain. The number of fixed replacement differences between A. thaliana and A. lyrata AP3 genes, however, is lower than observed synonymous fixed differences. The core region has 6 replacement and 14 synonymous differences, while the noncore region has 5 replacement and 10 synonymous changes. Contingency tests reveal that the relative ratio of within-species replacement to synonymous differences in the core domain is not significantly different when compared to differences between species (P = 0.238). The noncore region, however, contains an excess of intraspecific replacement polymorphisms compared to levels of between-species differences (P = 0.009).

The PI gene also exhibits an excess in levels of intraspecific replacement to synonymous polymorphisms in both functional domains of the gene. Both core and noncore regions have 6 replacement and 2 synonymous changes. The partitioning of the fixed differences, however, differs between core and noncore domains. Comparison of the core region of PI between A. thaliana and A. lyrata reveals only 1 replacement difference and 8 synonymous changes. In contrast, the noncore region also has 8 synonymous differences, but 10 replacement differences are observed between PI genes of these two species. For the noncore region, levels of intraspecific polymorphisms are correlated with interspecific replacement changes (P = 0.252). The core region of PI, however, displays low levels of between-species replacement difference (1/9) and an excess of within-species replacement polymorphisms (6/8), and this difference is significant (P = 0.014).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Variation at floral regulatory gene loci:
The APETALA3 and PISTILLATA loci of A. thaliana play central roles in floral organ development (JACK et al. 1992 ; GOTO and MEYEROWITZ 1994 ). Assessment of the levels of variation at AP3 and PI, as well as previous work on the floral meristem identity gene CAULIFLOWER (PURUGGANAN and SUDDITH 1998 ), indicates that these developmental regulatory genes possess appreciable levels of nucleotide variation. The overall estimates of intraspecific variation at these two organ identity genes ( = 0.005–0.0065) are comparable to the estimate for CAULIFLOWER ( = 0.0070). The levels of diversity of these three regulatory genes are also similar to nucleotide variation estimates for two structural loci (ChiA and Adh) in Arabidopsis (see Table 3).

Population subdivision and expansion of A. thaliana:
The extent and patterning of variation in loci are governed by the interplay of selective and demographic processes that shape the diversity of genes and genetic pathways. Evolutionary forces that affect the entire genome leave similar patterns across different unlinked loci and provide information on population history, structure, and dynamics. The specific details of population structuring in this plant species are crucial to understanding the nature of the evolutionary forces that shape diversity at these floral regulatory loci.

A recent study investigated variation in three nuclear loci within and between Arabidopsis populations using restriction site variation (BERGELSON et al. 1998 ). Comparison of this study with sequence data on AP3 and PI as well as three previously studied, unlinked loci—the CAL (PURUGGANAN and SUDDITH 1998 ), ChiA (KAMABE et al. 1997 ), and Adh (HANFSTINGL et al. 1994 ; INNAN et al. 1996 ) genes—permits us to evaluate common patterns of variation that presumably arise from their shared evolutionary histories within the A. thaliana genome. Table 3 summarizes three features characteristic of most Arabidopsis genes that have been thus far analyzed: (1) excess of low-frequency nucleotide polymorphisms, (2) excess of within-species replacement site variation, and (3) evidence for allelic dimorphism.

For both the AP3 and PI floral regulatory genes, the Tajima and Fu and Li tests reveal significantly negative D estimates, indicating a bias toward rare polymorphisms at these loci. A significant excess of singleton changes is also observed for ChiA (KAMABE et al. 1997 ), while CAL shows negative D* statistics that are significant at the 10% level (P < 0.057 in the Fu and Li test; PURUGGANAN and SUDDITH 1998 ). Excess in low-frequency alleles can arise from hitchhiking effects that accompany selective sweeps, which reduce linked sequence variation at specific loci (AQUADRO 1997 ). The regulatory genes AP3 and CAL (PURUGGANAN and SUDDITH 1998 ), however, as well as the structural loci ChiA (KAMABE et al. 1997 ) and Adh (INNAN et al. 1996 ), all possess intermediate frequency polymorphisms that differentiate allele classes; this variation would presumably have been eliminated by genetic hitchhiking during species-wide fixation of selected sites.

The excess in low-frequency polymorphisms may be explained by the background selection hypothesis (CHARLESWORTH et al. 1993 ), which suggests that rare variants may be present in population samples as a result of linkage to deleterious mutations. This effect should be marked in regions of low recombination or in primarily selfing species such as Arabidopsis. The background selection hypothesis, however, does not explain the excess in within-species replacement polymorphisms common to most of the Arabidopsis nuclear loci that have been examined (see Table 3).

Population subdivision and/or recent population expansion are mechanisms that may explain, however, both the excess in low-frequency allelic variation for Arabidopsis nuclear loci and the excess of within-species replacement polymorphisms (AQUADRO 1997 ). Despite occasional cross-pollination, low outcrossing rates in A. thaliana may have resulted in population subdivision across the species range as a result of limited gene flow between inbred subpopulations (ALLARD et al. 1968 ; CHARLESWORTH et al. 1997 ; NORDBORG 1997 ). Inbreeding plant populations generally exhibit low within-population heterozygosities, given the low effective population sizes of primarily selfing organisms (ALLARD et al. 1968 ). Reduced levels of within-population variation have been observed for allozyme (ABBOTT and GOMES 1989 ), microsatellite (TODOKORO et al. 1996 ; KUITTINEN et al. 1997 ), and mitochondrial and nuclear restriction site studies (BERGELSON et al. 1998 ) in Arabidopsis populations. Inbreeding plants, however, also display elevated levels of between-population differentiation as alternate genotypes are fixed in different populations by both drift and selection (ALLARD et al. 1968 ). The excess of low-frequency polymorphisms observed in this species-wide survey of AP3 and PI nucleotide variation in A. thaliana may result from overdispersed sampling of distinct, inbred populations.

Differentiated, inbred populations of low effective population size would also explain the widespread occurrence of excess intraspecific replacement polymorphisms in nuclear loci of A. thaliana. The McDonald-Kreitman tests indicate that both AP3 and PI, as well as CAL (PURUGGANAN and SUDDITH 1998 ) and ChiA (KAMABE et al. 1997 ), have significantly high levels of intraspecific replacement polymorphisms compared to levels of interspecific divergence. The nearly neutral theory suggests that the intensity of purifying selection is relaxed and slightly deleterious mutations may be fixed at a higher rate in small populations (OHTA 1992 ). If the ecotypes sampled represent distinct, inbred populations of small Ne, then this would suggest that high levels of observed species-wide intraspecific replacement polymorphisms result from slightly deleterious mutations that are confined to individual ecotype populations.

Recent, rapid population expansion (CUMMINGS and CLEGG 1998 ) could also account for the excess in low-frequency and replacement site polymorphisms observed in A. thaliana. It is likely that both population subdivision and expansion have played roles in structuring the molecular diversity within this selfing plant species. Because population subdivision is associated with selfing plant species, one may expect that some of the features observed in A. thaliana may be shared by other inbreeding plant groups. There are indications that at least some of the features of nucleotide variation found in Arabidopsis characterize nuclear gene diversity in other selfing plants as well. The A. gemmifera Adh gene, for example, also displays a significant excess of within-species low-frequency polymorphisms and intraspecific replacement variation (MIYASHITA et al. 1996 ). Population subdivision in this selfing species has also been invoked to explain the patterning of diversity observed at this locus. The Adh locus from the self-fertilizing H. vulgare also displays a significant excess of within-species replacement polymorphisms and a greater (though not significant) frequency of singleton variation (CUMMINGS and CLEGG 1998 ).

Dimorphic variation in Arabidopsis nuclear genes:
Four of the five Arabidopsis genes examined display varying degrees of allelic dimorphism (INNAN et al. 1996 ; KAMABE et al. 1997 ; PURUGGANAN and SUDDITH 1998 ). The observed dimorphism for most genes is based on only a few sites; in the AP3 gene, for example, class A and B alleles are differentiated by only four linked polymorphisms in intron 4. The widespread occurrence of allelic dimorphism in Arabidopsis nuclear loci has been ascribed to either introgression from related species or the breakdown of barriers between two previously isolated populations (HANFSTINGL et al. 1994 ; KAMABE et al. 1997 ).

It is unclear what mechanisms have contributed to the continued maintenance of polymorphisms that differentiate allelic classes in A. thaliana nuclear genes. Association analysis reveals that the four polymorphisms that differentiate the AP3 allele classes are in complete linkage disequilibrium with each other. In CAL, the polymorphic sites that differentiate two allelic classes are spread out over a 260-bp region and include a distinctive replacement polymorphism that may account for phenotypic variation in floral homeotic function. Linkage among sites, coupled with the low outcrossing rates for A. thaliana, may have led to the persistence of these sites in linkage disequilibrium.

Selective forces may also operate to maintain these linked polymorphisms. The background selection model suggests that balancing selection in selfing plants such as A. thaliana would lead to linkage among neutral variants and selected sites (NORDBORG et al. 1996 ). A similar pattern should also be observed under local selection in subdivided populations (CHARLESWORTH et al. 1997 ). Given their location within introns, however, the selective advantages of the linked changes responsible for the dimorphism at AP3 are not immediately apparent. Studies on evolution of intron sites suggest that some selective pressures related to the formation of pre-mRNA hairpin structures may be involved in gene regulation (KIRBY et al. 1995 ; PARSCH et al. 1997 ). RNA secondary structure analysis indicates that a hairpin associated with these AP3 polymorphic sites may possess different melting temperatures in the two allele classes (M. D. PURUGGANAN, unpublished observations). Moreover, molecular studies in the related MADS-box floral homeotic gene AGAMOUS reveal that regulatory sites within introns may modulate expression of this regulatory gene (SIEBURTH and MEYEROWITZ 1997 ). No comparable studies for AP3 have been reported, although it is possible that cis-acting regulatory sequences may also be present in the introns of this floral homeotic gene.

Evolutionary dynamics of functional domains within APETALA3 and PISTILLATA:
The distribution of replacement variation between distinct functional domains of APETALA3 and PISTILLATA suggests that different regions of these regulatory proteins experience contrasting evolutionary pressures. These differing evolutionary forces between domains are reflected in the distribution of replacement and synonymous changes between gene regions (TEMPLETON 1996 ). On the basis of a comparison of the patterning of coding region variation among several genes, the expectation for A. thaliana is (1) an excess of within-species replacement polymorphisms as a result of an increased tolerance for slightly deleterious mutations in small, inbred populations, and (2) a lower ratio of interspecific replacement-to-synonymous differences arising from purifying selection that prevents fixation of polymorphisms across species.

Contingency analyses can incorporate functional information and partition the mutations between distinct gene domains (RIECHMANN and MEYEROWITZ 1997 ). We can examine whether the pattern of replacement to synonymous variation is similar between the core and noncore domains of A. thaliana AP3 and PI genes (see Table 2). The patterns of replacement and synonymous differences between A. thaliana and A. lyrata indicate that both core and noncore domains of APETALA3 are constrained against amino acid changes. In these two domains, levels of replacement differences are lower than synonymous ones, suggesting that purifying selection prevents the fixation of amino acid polymorphisms.

This trend is reversed, however, in the distribution of within-species polymorphisms. The noncore region of AP3 has 15 replacement and only 5 synonymous polymorphisms, which suggests that this domain can tolerate a high level of possibly slightly deleterious amino acid changes. Contingency tests on levels of replacement and synonymous changes in the AP3 noncore region reveal the expected (standard pattern) of significant excess in intraspecific replacement polymorphisms. The AP3 core region, however, shows a lower level of replacement to synonymous polymorphisms (5 replacements to 4 synonymous); this ratio is not significantly different from observed interspecies differences. Given the low overall numbers of polymorphisms and differences in the AP3 core region, the contingency test applied may have restricted power. Otherwise, this test does indicate that the core region of AP3, which performs central functions in this floral regulatory protein, is less tolerant of replacement mutations than the noncore domain.

This contrast in evolutionary dynamics between core and noncore regions is also exhibited by the PISTILLATA protein-coding region. Contingency testing suggests that in PI it is the core region that is behaving according to the expected standard pattern—high levels of intraspecific replacement polymorphisms and reduced levels of interspecific amino acid change. The noncore region, however, deviates from this expected pattern and indicates that a large number of replacement substitutions have been fixed in the divergence between the A. thaliana and A. lyrata PI locus. The difference between the pattern of interspecific divergence in core and noncore regions is significant (P = 0.042 using Fisher's exact test).

The interspecific amino acid changes in the PI noncore region are centered primarily in the C-terminal region, which previous molecular evolutionary analyses have indicated is the most rapidly evolving structural domain of plant MADS-box proteins (PURUGGANAN et al. 1995 ). This high level of amino acid change could arise from a relaxation in the functional constraint at PISTILLATA after the separation of these two Brassicaceae species or adaptive fixation of selectively favored amino acid changes. AP3 and PI proteins form a heterodimer necessary for nuclear localization and DNA-binding (RIECHMANN et al. 1996 ), and the differences in the evolutionary dynamic between domains may arise as a result of some degree of functional complementation between proteins. It should be noted that there are no dramatic differences in the floral morphologies between A. thaliana and A. lyrata, although the petals of the latter are about twice as large (3–4 mM in A. thaliana vs. 7–10 mM in A. lyrata). The growth trajectories of petals and stamens should also differ between the two taxa as the selfing syndrome evolved in A. thaliana.

The molecular population genetics of the floral homeotic genes APETALA3 and PISTILLATA provide insights into both the population structure of A. thaliana and the dynamics of regulatory gene variation. Population structuring could account for the excess of both singleton variation and replacement polymorphisms in species-wide surveys, and the subdivision of A. thaliana populations determines, in part, the fate of mutational variation at these regulatory genes. The pattern of variation indicates that functional domains evolve differently from one another within these two floral regulatory genes, which suggests that members of the floral developmental pathway are subject to distinct evolutionary forces.

	ACKNOWLEDGMENTS

We would like to acknowledge Ed Buckler, Katy Simonsen, and Elizabeth Friar for a critical reading of this paper and helpful discussions and two anonymous reviewers for suggested improvements. We also gratefully acknowledge Elliot Meyerowitz for providing us with the original AP3 and PI genomic sequences. This work was funded with a grant from the U.S. Department of Agriculture National Research Initiative Plant Genetic Mechanisms Grant 97-35301-4688 and an Alfred P. Sloan Foundation Young Investigator Award in Molecular Evolution to M.D.P.

Manuscript received May 12, 1998; Accepted for publication October 30, 1998.

	LITERATURE CITED

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