* Department of Biology, Duke University
School of Botany, University of Melbourne, Victoria, Australia
Correspondence: E-mail: marcy{at}duke.edu.
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Abstract |
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Key Words: self-incompatibility S-locus S-RNase
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Introduction |
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In the SSI system of Brassica, pollen specificity is vested in a small, cysteine-rich protein embedded in the outer coat of pollen grains (Schopfer, Nasrallah, and Nasrallah 1999) and pistil specificity resides in a receptor protein kinase bound in the plasma membrane of epidermal stigmatic cells (Takayama et al. 2001). A functional ribonuclease (S-RNase) determines pistil specificity in the solanaceous GSI system (Lee, Huang, and Kao 1994; Murfett et al. 1994). S-RNase genes also cosegregate with S-loci within the Rosaceae (Sassa, Hirano, and Ikehashi 1992) and Scrophulariaceae (Xue et al. 1996). Phylogenetic analyses of the sequence and structure of the S-RNase genes indicate a common origin of GSI in these distantly related families (Igic and Kohn 2001; Steinbachs and Holsinger 2002). Identification of the gene, provisionally designated pollen-S, that controls pollen specificity in S-RNasebased GSI systems remains a prime objective. Here we assess a candidate for pollen-S by analyzing the pattern of nucleotide variation.
Recombination Suppression Within the S-Locus
Suppressed recombination between the pollen and pistil genes is considered essential to SI function, as crossing-over would presumably generate a recombinant haplotype that fails to reject the specificity expressed by its own pollen. In the SSI system of Brassica, the extensive structural differences observed among segregating S-alleles (Boyes et al. 1997; Suzuki et al. 1999; Nasrallah 2000) would appear to preclude the generation of functional haplotypes by conventional crossing-over in a region spanning perhaps hundreds of kilobases.
Recent studies have greatly improved the resolution of the S-locus region in S-RNase-based GSI (Li et al. 2000; Dowd et al. 2000; Lai et al. 2002). As in the SSI system of Brassica (Suzuki et al. 1999), the abundance of repetitive sequences in regions flanking the pistil-expressed S-RNase gene in the Solanaceae (Coleman and Kao 1992; Matton et al. 1995), Rosaceae (Ushijima, Sassa, and Hirano 1998), and Scrophulariaceae (Lai et al. 2002) suggests suppressed recombination. In solanaceous species, the S-locus lies in a centromeric region in which recombination appears to be suppressed over at least one megabase and possibly several (McCubbin and Kao 1999; McCubbin, Wang, and Kao 2000). In Brassica rapa, the S-locus region is gene-rich, containing approximately one expressed gene per 5.4 kb (Suzuki et al. 1999). If gene density within the S-RNase-based GSI S-locus is also of this order, hundreds or even thousands of genes may exist for which recombination with S-RNase would be undetectable in samples of practicable size. Indeed, McCubbin, Wang, and Kao (2000) have isolated several pollen-expressed genes near S-RNase in Petunia inflata (Solanaceae), and Lai et al. (2002) predicted 11 genes within a 63-kb region surrounding a functional S-RNase allele of Antirrhinum hispanicum (Scrophulariaceae). A number of floral traits involved in pollination biology have been shown to be linked to the Lycopersicon hirsutum S-locus (Bernacchi and Tanksley 1997).
Candidates for pollen-S
Several genes isolated from the S-locus region of S-RNase-based GSI systems exhibit characteristics considered fundamental to pollen-S: pollen-specific expression, complete allelic linkage disequilibrium with S-RNase (a distinct restriction fragment length pattern for each S-haplotype examined), and no detectable recombination with S-RNase. These include 48A in Nicotiana alata (Solanaceae; Li et al. 2000), 13 genes in P. inflata (Solanaceae; McCubbin, Zuniga, and Kao 2000), and F-box protein genes in species of Prunus (Rosaceae; Entani et al. 2003; Ushijima et al. 2003;).
A direct assessment of the involvement of a gene in SI function might entail examining whether transgenic plants show a change in SI expression (for example, Lee, Huang, and Kao 1994; Schopfer, Nasrallah, and Nasrallah 1999). However, with respect to the pollen-S gene in an S-RNase-based system, the analysis of such experiments may not be straightforward, as it would rely on an interpretation of the complex pollination phenotype seen in self-compatible mutant plants that carry an extra copy of all or part of an S-allele (Golz, Clarke, and Newbigin 1999; Golz et al. 2001). It is not known whether an appropriately engineered pollen-S gene construct would produce this phenotype when introduced into the genome of a self-incompatible host plant, or indeed whether this phenotype is displayed by all plants with an extra copy of an S-allele. Moreover, a transgenic approach may be impossible in some species, either because genetic transformation is far from routine or because plants must be grown for long periods before starting to flower.
We here present an analysis of nucleotide sequence variation designed to assess whether 48A, derived from N. alata (Li et al. 2000), is a likely candidate for the determinant of pollen specificity in the S-RNase-based system of GSI. Results of a parallel analysis of the SSI system of Brassica, for which both the pollen and stigmatic determinants are known, appear to verify our interpretations.
First, we applied maximum-likelihood (ML) methods developed by Yang and colleagues to identify targets of positive selection in both systems. Second, we examined whether the pollen-expressed gene and the S-locus have had a common evolutionary history. Indications of recombination include departures from proportionality of the lengths of corresponding branches in the genealogical trees of the two genes and differences in the numbers of segregating sites. Because these indicators would be influenced by the acceleration by balancing selection of substitution at specificity-determining sites (Maruyama and Nei 1981; Takahata 1990; Sasaki 1992), we based our inferences of genealogical history on the pattern of synonymous or untranslated variation in the partition remaining after the removal of all sites that showed even weak indications of positive selection.
Our analysis confirmed that the determinant of pollen specificity in the Brassica SSI system fulfills the expectations of positive selection and common history with the pistil gene. In contrast, 48A showed no evidence of positive selection; further, the highly significant reduction in the number of segregating neutral mutations relative to S-RNase suggests historical recombination between the genes. We conclude that 48A may not correspond to the determinant of pollen specificity in the S-RNase-based GSI system.
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Materials and Methods |
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Our analysis of variation within the Brassica SSI system examined a total of 15 SRK and 26 SP11 class I alleles, including 11 haplotypes for which both the pistil and the pollen components were available (table 2). We restricted our study to the highly polymorphic ectodomain region of SRK, which determines pistil specificity, and the entire coding region of SP11, excluding the signal sequence. After adjustment of sequence length to permit comparison of all 11 two-locus haplotypes, our data set spanned 421 codons of exon 1 of SRK and 80 codons of SP11.
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Yang and colleagues developed a ML framework for characterizing the process of substitution at specific codons or sites within one or more genomic regions, given a specified genealogy (Goldman and Yang 1994; Yang 1996; Nielsen and Yang 1998; Yang et al. 2000; Yang and Swanson 2002). Aspects of the substitution process examined include nonsynonymous/synonymous substitution rate ratio ( = dn/ds), transition/transversion substitution rate ratio (
), codon frequencies (
), rate of substitution, and the shape parameter of gamma-distributed variation in substitution rate among sites. To identify possible targets of positive selection, individual codons can be assigned to one of a number of classes that differ, for example, with respect to the relative rates of synonymous and nonsynonymous substitution within a gene (Nielsen and Yang 1998; Yang et al. 2000). An alternative to this random-sites model is the fixed-sites model (Yang 1996; Yang and Swanson 2002), in which groups of sites may be defined by the user on the basis of prior information, including protein structure and function. This implementation permits comparisons of genomic regions between or within genes with respect to various aspects of the evolutionary process. Within this ML framework, comparisons of log-likelihood ratios provide the basis for statistical testing of nested evolutionary hypotheses (summarized in Table 2 of Yang 1996; Table 2 of Yang et al. 2000; Table 1 of Yang and Swanson 2002).
We used the CODEML module of PAML (Yang 1997) to conduct our codon-based analyses, which include random-sites models for the identification of targets of positive selection and fixed-sites comparisons among designated partitions. We used the BASEML module to compare the 3' UTR of 48A and third codon position sites within S-RNase and 48A.
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Excluding Possible Targets of Positive Selection |
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Model 3 of Yang et al. (2000) estimates for each of a specified number of classes a value of the relative rate of nonsynonymous to synonymous substitution ( = dn/ds). Each codon triplet is assigned to the class for which the posterior probability of its having come from the class is highest. At one extreme, one might set the number of classes equal to the number of codon sites and designate any site with an
value exceeding unity as a possible target of positive selection. In practice, we began with a one-class model (Model 0) and incremented the number of classes until the estimated
values for all codons appeared to stabilize, with any additional class assigned an
value identical to that of an existing class. We then adopted the first model (lowest number of classes) that gave the stable values, designating the union of all classes with
values greater than unity as the positive selection partition and the complementary set of sites as the conservative evolution partition. Because our primary objective is to infer evolutionary history from the pattern of variation within the conservative partition, the positive selection partition may well include sites for which evidence of positive selection is weak.
Bootstrap Test of Base or Codon Composition
To examine differences in codon frequencies between partitions, Yang et al. (2000) suggested a likelihood-ratio test comparing Models B and C, which are described as differing only with respect to permitting unequal codon frequencies between partitions. However, the models are not in fact nested. In each model, estimates of the codon frequencies are not optimized together with the other parameters but rather are assigned as the products of the observed base frequencies across codon positions (F3 x 4) or as the observed codon frequencies (F61; see Goldman and Yang 1994). Further, none of the models considered accounts for sampling error of the observed base or codon frequencies.
We developed a bootstrapping procedure to address whether base or codon frequencies differ significantly among partitions. For two partitions comprising nx and ny nucleotide or codon sites, we determined for each site a k-dimensional vector representing the observed proportions of the possible states (k = 4, 61 for nucleotides and codons, respectively). We then generated random partitions of the nx + ny sites by assigning nx sites, sampled with replacement, to one group and ny sites to the other. For each resampled set, we determined the distance between the groups from
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Expression (1) was originally proposed as an index of distance between genetic samples obtained from different populations (Nei 1987, chapter 9). For xi and yi following Gaussian distributions, (1) follows a 2 distribution. Nei (1987) noted that the simple Euclidean distance between partitions has the undesirable property that completely complementary sets of bases or codons show only moderate distance. The weighting imposed by the denominator of equation (1) removes this feature.
We restricted likelihood comparisons to models that specified identical or different codon frequencies across partitions, in accordance with the results of the bootstrapping tests. Our analyses used the F3 x 4 model, which determines codon frequencies as products of the base frequencies observed at the three codon positions.
A Test of Relative Numbers of Segregating Sites
For individual partitions within haplotypes, we estimated the total number of neutral substitutions which have occurred since the most recent common ancestor (MRCA). We developed the procedure outlined in the Appendix to compare the relative numbers of substitutions between two partitions within the sampled haplotypes. Our null hypothesis proposes absolute linkage and equal rates of substitution between partitions. Under this hypothesis, two partitions share a common genealogical history, with differences in substitution number attributed entirely to random events. We compared relative numbers of synonymous substitutions between coding regions or substitutions between third codon positions of one gene and a noncoding region of another gene.
In accordance with the null hypothesis, we used PAUP* 4.0b10 to obtain an ML estimate of the joint genealogy from concatenated partitions and the CODEML module of PAML to estimate per-site substitution numbers for each branch of the joint genealogy. We then summed these estimates over the entire genealogy and multiplied by the number of sites to obtain the total number of substitutions occurring since the MRCA of the haplotypes (the number of segregating sites under the infinite sites model).
Under the standard assumption that the number of substitutions occurring within a specified time period follows a Poisson distribution, we tested whether the numbers of segregating sites in two partitions are consistent with a common value for the Poisson parameter. Rejection of the null hypothesis might reflect (1) different rates of substitution between partitions with a shared genealogical history or (2) identical substitution rates but different genealogies. In accordance with the first explanation, we estimated the Poisson parameter for each partition separately under the joint genealogy and used likelihood ratio tests to determine whether the parameters differ between partitions. In accordance with the second explanation, we obtained an ML genealogy for each partition separately and used estimates of the numbers of segregating sites to determine ML estimates of the Poisson parameters.
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Results |
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Positive Selection and Conservative Evolution Partitions
Figure 1 indicates the amino acid residues corresponding to codon sites assigned to the positive selection partition of S-RNase. Stabilization of estimates and site assignments occurred at six classes, with no new
values or site assignments indicated even in a model permitted 16 classes (table 3). Estimated
values exceeded unity for two of the six classes, comprising 52 of the 214 codons studied. In contrast, no sites within 48A were identified as possible targets of positive selection, under any assignment of class number from one through seven. Figure 1 compares the sites assigned to the positive selection partition of S-RNase with regions HVa and HVb, designated as hypervariable by Ioerger et al. (1991), and with regions PS1, PS2, PS3, and PS4, in which the sliding window analysis of Ishimizu et al. (1998) detected an excess of nonsynonymous substitutions in rosaceous S-alleles.
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Base composition and codon usage in the coding region of 48A differed highly significantly from those in S-RNase. In particular, the first and second codon positions of 48A exhibited a very strong bias toward A and G in contrast with the third codon position and 3' UTR, and the third codon position was more G/C-rich than the 3' UTR. Differences in base composition between the 3' UTR of 48A and the third codon positions of either S-RNase partition were nonsignificant.
Every comparison involving the conserved partition of SP11 showed highly significant differences in both base composition and codon usage. Both aspects reflect the predominance of Cys residues (codons TGT and TGC).
Variation Within Pistil-Expressed S-RNase
We conducted fixed-site comparisons (Yang and Swanson 2002) of the positive selection and conservative evolution partitions of S-RNase (fig. 1). Model B of Yang and Swanson (2002) is described as nested within Model C, differing only in permitting different codon frequencies between partitions. However, our observation of a higher estimated likelihood of B confirms that B is not in fact nested within C.
Our bootstrap test of base composition (see Materials and Methods) offers an alternative method for comparing codon usage between genes or regions within genes. This analysis indicated significant differences between partitions at the first codon position but nonsignificant differences in codon usage (table 5). In accordance with the significant result, we adopted model F3 x 4 and permitted different codon frequencies between partitions in our fixed-site comparisons of the positive selection and conservative evolution partitions of S-RNase.
Table 6 summarizes the results of likelihood ratio comparisons among models. A fixed-site comparison between the positively selected and conserved partitions of S-RNase indicated significant differences in and
(Model C versus Model E: P = 5.7 x 1029). A model that treated the two partitions as separate data sets and permitted differences between partitions in substitution rate, codon frequencies,
, and
had significantly higher likelihood than one that assumed a common genealogy, indicating rejection of proportionality of lengths of corresponding branches between partitions, though at a much lower level of significance (E versus F: P = 0.01). This most general model (F) indicated a 4.3-fold higher substitution rate in the positive selection partition.
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Variation Within Pollen-Expressed 48A
We conducted a fixed-site comparison between third codon positions and the 3' UTR of 48A. Because our null hypothesis asserts absolute linkage between S-RNase and 48A, we specified the S-RNase topology (fig. 2) for these partitions within 48A. We also compared the partitions under the topology estimated from variation at 48A alone.
Under either topology, comparison among models that assumed gamma-distributed substitution rates among sites within partitions and permitted different base frequencies between partitions indicated no significant differences between partitions in the shape parameter, rate of substitution, , or lengths of corresponding branches.
Under the topology determined from 48A alone (fig. 3), a model that permitted differences between partitions in base composition and overall substitution rate but not in appeared to fit the data best. This model indicated an L-shaped distribution of substitution rate among sites within partitions (gamma-distribution shape parameter = 0.061). The log likelihood (lnL) of this best-fit model under the 48A tree considerably exceeded that under the S-RNase tree (2
lnL = 2(541.5 + 564.4) = 45.8). Because the topology hypotheses are not nested, this value cannot be directly tested against a
2 distribution; however, its magnitude provides another indication that the 48A and S-RNase regions may have had distinct evolutionary histories. We tested this issue directly by comparing the numbers of segregating synonymous substitutions in the two genes (see next section).
Between-Gene Comparisons
We first made codon-based comparisons between the entire coding region of 48A and the conservative evolution or positive selection partition of S-RNase. We then compared the 3' UTR of 48A to the third codon positions of each of the S-RNase partitions. Each comparison used the topology estimated from the particular sequences being compared.
Codon-Based Analysis of S-RNase and 48A
We detected highly significant differences in substitution rate between the conservative evolution partition of S-RNase and the coding region of 48A (P = 1.5 x 1057). A comparison of models that allowed differences in codon frequency () and substitution rate between partitions indicated nonsignificant differences in
and
. A test of proportionality of the numbers of substitutions in the S-RNase and 48A partitions along corresponding branches indicated nonsignificant differences.
A comparison between the positive selection partition of S-RNase and the coding region of 48A gave similar results: highly significant differences in substitution rate (P = 1.2 x 10117) and proportional branch lengths. Although the ML estimates of appeared to differ strongly between genes (1.6 for S-RNase and 0.3 for 48A), the difference was not significant. The low level of polymorphism within the coding region of 48A (eight variable nucleotide sites among 261) suggests limited statistical power.
A general model, permitting differences between partitions in ,
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, and substitution rate, indicated that substitution within the conserved partition of S-RNase proceeds at a rate nearly 19-fold higher than in the coding region of 48A, with the positive selection partition of S-RNase showing even greater acceleration (nearly 80-fold).
3' UTR of 48A and Third Codon Positions of S-RNase
We compared the more polymorphic 3' UTR of 48A to third codon positions of the conserved partition of S-RNase. Comparison of models that assumed uniform substitution rate among sites within partitions and identical values between partitions indicated highly significant differences in substitution rate between partitions (P = 3.5 x 1038). Corresponding branch lengths of the S-RNase and 48A partitions showed significant departures from proportionality, suggesting differences in evolutionary history between the genes.
Comparison of the 48A 3' UTR to the third codon positions of the positive selection partition of S-RNase gave similar results. We found a highly significant (P = 2.7 x 1044) acceleration of substitution rate in S-RNase, even at the third codon position, and confirmed significant nonproportionality of corresponding branch lengths.
Relative Numbers of Substitutions in S-RNase and 48A
Comparisons of the numbers of segregating sites in S-RNase and 48A (table 7) indicated highly significant departures from expectation under the hypothesis of absolute linkage and equal rates of substitution between genes.
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Alternatively, the apparent deficiency of segregating sites in 48A may indicate a shallower genealogy, reflecting historical recombination. Comparison of the numbers of segregating sites estimated from independent genealogies for S-RNase and 48A indicated 18.7-fold more synonymous substitutions in S-RNase. This result suggests a considerable reduction in total tree length under the assumption that synonymous substitution proceeds at comparable rates in the two genes.
SSI Sequences
Fixed-site comparisons between the positively selected and conserved partitions of the pistil (SRK) and pollen (SP11) components of the Brassica SSI system confirmed significantly higher and substitution rate in the positively selected partition (table 6). The lengths of corresponding branches between partitions within SRK departed significantly from proportionality.
To examine further the nature of the nonproportionality of branch lengths, we compared third codon positions between the positive selection and conserved partitions of SRK. This analysis again indicated a highly significant, though smaller, acceleration in substitution rate. However, departures from proportionality of corresponding branch lengths became nonsignificant (table 6), consistent with the absence of recombination within the pistil gene.
Comparison of the positive selection partitions of the pollen (SP11) and pistil (SRK) genes indicated a significantly higher rate of substitution in SP11 (P = 3.7 x 1022) and significant departures from proportionality of corresponding branches. In contrast, no significant differences were detected between the conserved partitions of these genes. Although the finding of apparent proportionality of branch lengths is in accordance with our expectation of absolute linkage between the genes, it may also reflect low statistical power in comparisons involving the small conserved partition of SP11, which shows very little variability (six of 31 codons).
Restricting the between-gene comparison to the third codon position across the entire coding region confirmed a higher substitution rate in SP11. However, nonproportionality of corresponding branch lengths remained significant, albeit at a lower level (table 6). Comparison of the third positions of the positive selection partitions between the pollen and pistil genes gave similar results, including significant nonproportionality of corresponding branch lengths. Comparison of the third codon positions in the conservative evolution partitions indicated no significant differences between the genes.
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Discussion |
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Our study of the pattern of nucleotide variation at 48A (Li et al. 2000) suggests that this candidate gene may not correspond to pollen-S. Three lines of evidence support this conclusion: (1) absence of indications of positive selection, (2) departures from the pattern exhibited by the pollen component of the system of SSI expressed in Brassica, and (3) apparent historical recombination with S-RNase, the pistil component of this GSI system.
Distinguishing Between Selection and History
Our assessment addressed whether the candidate for pollen-S has been subject to positive selection and shows absolute linkage to the determinant of pistil specificity, both features considered essential to the function of the determinant of pollen specificity. Our examination of these two aspects used complementary regions within S-locus genes, the positive selection and conservative evolution partitions. Because we base inferences of historical recombination on the relative numbers of substitutions in the pistil and pollen genes, we require a view of history that is largely free from changes in substitution rate induced by selection.
Self-incompatibility engenders an intense form of balancing selection (Vekemans and Slatkin 1994), reflecting the transmission advantage of pollen genes that express rarer specificities. Balancing selection among functionally distinct specificities maintains large numbers of specificities and greatly expands coalescence times (Takahata 1990). In accordance with expectation, S-loci typically show extraordinary polymorphism (Lawrence 2000); furthermore, genes that determine pistil specificity in model systems of both GSI and SSI show very ancient divergence among lineages (Ioerger et al. 1990, Dwyer et al. 1991), in excess of 30 Myr in both the solanaceous GSI system (Ioerger, Clarke, and Kao 1990) and the Brassica SSI system (Uyenoyama 1995). Whereas balancing selection accelerates substitution at specificity-determining sites (Maruyama and Nei 1981; Takahata 1990; Sasaki 1992), the rate of substitution of neutral variants reflects only the rate at which they arise, irrespective of linkage to sites subject to selection (Birky and Walsh 1988).
Hughes and Nei's (1988) seminal approach to the detection of positive selection used information obtained from crystal structure analyses of MHC molecules to identify a candidate target region and documented significantly higher ratios of nonsynonymous to synonymous substitutions in that region compared to other regions of the molecule. Ishimizu et al. (1998), in their sliding window analysis of rosaceous S-RNase genes, inverted this approach, identifying regions that showed significant excess nonsynonymous substitutions as candidates for targets of positive selection. We applied the Nielsen/Yang ML method (Nielsen and Yang 1998; Yang et al. 2000), which assigns individual codons to classes that differ with respect to the ratio of nonsynonymous to synonymous substitution rates.
To address the second objective, of assessing whether the pollen-expressed gene shares its evolutionary history with the pistil-expressed gene, we restricted our analysis to sites that showed no indications of positive selection. We assigned to the positive selection partition any site for which the posterior probability of having come from a class with a higher rate of nonsynonymous substitution than synonymous substitution ( = dn/ds > 1) exceeded that for other classes. We accepted even low posterior probabilities, because our intention was to make a conservative determination of the complementary set of sites. Even within the conservative evolution partition, we further restricted consideration whenever possible to synonymous variation or variation at third codon positions.
We interpreted significant differences in numbers of segregating sites and departures from proportionality of corresponding branch lengths in the genealogies of the pistil- and pollen-expressed genes as evidence of historical recombination. Recombination with specificity-determining sites would reduce the expansion of coalescence times at neutral sites induced by balancing selection (Hudson and Kaplan 1988). Separate genealogical histories of different regions within haplotypes can also cause apparent differences in substitution rates along genealogical branches that would, in the absence of recombination between the regions, correspond to equal time intervals. Actual differences between regions in substitution rate may of course also account for differences in sequence divergence.
Selection Within the S-Locus Region
Figures 1, 4, and 5 indicate the positive selection and conservative evolution partitions of the pistil gene (S-RNase) of the solanaceous GSI S-locus and of the pistil (SRK) and pollen (SP11) components of the Brassica SSI S-locus. For each gene, fixed-site comparisons confirmed significantly greater values and higher overall rates of substitution in the positive selection partition than in the conservative evolution partition (table 6). We detected no indication of positive selection within 48A.
Figure 1 shows the two regions (HVa and HVb) which Ioerger et al. (1991) designated as hypervariable within solanaceous S-RNase alleles and the four regions (PS1, PS2, PS3, and PS4) in which Ishimizu et al. (1998) detected an excess of nonsynonymous substitutions in rosaceous S-RNase alleles. Most of the sites assigned to a class under positive selection ( > 1) fall within these regions. The offset between PS4 and the three sites near the C-terminal with high posterior probability (P > 0.95) of assignment to a positive selection class may reflect some ambiguity, especially near the terminii, in inferring functional similarity or homology between the rosaceous S-alleles studied by Ishimizu et al. (1998) and solanaceous S-alleles.
Ida et al. (2001) have solved the three-dimensional structure of S-RNase Sf11 (N. alata), the first sequence shown in figure 1. All but one of the eight codons assigned with the highest posterior probability (P > 0.99) to a positive selection class fall within the HV and PS regions. The exceptional residue (Lys46, with numbering as shown in figure 1) lies on the surface of the Sf11 protein, a position that does not exclude its possible involvement in allelic interactions. Ida et al. (2001) reported two clefts near the active site for RNase catalysis that are large enough to act as substrate binding sites. Of the 13 amino acids that make up these clefts, only one (Pro93) was assigned to a positive selection class with high posterior probability (P > 0.95).
In contrast with S-RNase, no sites within 48A were assigned to the positive selection partition. Furthermore, our comparison of the relative numbers of segregating sites (table 7) indicates that 48A has experienced significantly fewer synonymous and third position substitutions than S-RNase. None of our various analyses detected evidence of positive selection in the pollen-expressed 48A gene, although low sample size (five) likely limited statistical power.
Although it lacks any indication of positive selection, the coding region of 48A shows unusual base composition, including very strong bias toward A and G in the first and second codon positions in comparison to the third position and 3' UTR. A bootstrap comparison confirmed significant differences in base composition between the third codon position and the 3' UTR as well (table 5). Although the function of 48A in the pollen grain is unknown, the high content of charged amino acids (30% Lys, 16% Asp, 14% Glu) and similarity to a group of equally hydrophilic proteins expressed by plant tissues undergoing some form of dessication (Thomashow 1999) suggest a possible role in preventing damage to pollen grain membranes. Mature Nicotiana pollen grains exist in an essentially dry state, reflecting a substantial withdrawal of water during the latter stages of development; upon hydration, a comparable amount of water flows back into the grain (Lush, Grieser, and Wolters-Arts 1998). In the absence of suitable protection, these fluxes of water into and out of the grain would cause cellular membranes to rupture. Another member of this group of proteins, COR15a, significantly increases freezing tolerance of plastids when overexpressed in Arabidopsis (Artus et al. 1996), possibly by associating with the membrane and preventing freeze-induced phase transitions (Steponkus et al. 1998). Like dessication, freezing damages plant tissues, initially through the removal of water and then, as tissues thaw, by the entry of water back into cells.
Consideration of the evolutionary conflicts that arise between the pistil or pollen components of SI, even under absolute linkage, suggests that the origin of new S-specificities may begin with a change in the pollen component (Uyenoyama and Newbigin 2000; Uyenoyama, Zhang, and Newbigin 2001), although other possibilities exist (Matton et al. 1999; 2000). Under any scenario, the maintenance of the highly specific recognition reaction between pollen and pistil would appear to require comparable rates of nonsynonymous change in the two components. Unlike 48A, the pollen component of the Brassica S-locus bore out the expectation of positive selection. Almost all sites within SP11 were assigned to the positive selection partition, with the exceptions corresponding primarily to highly conserved cysteine residues (fig. 5). Moreover, the overall rate of substitution in the positive selection partition of the pollen gene significantly exceeded that of the pistil gene (SRK P versus SP11 P in table 6).
Evidence of Historical Recombination
Hughes (2000) compared closely related class II major histocompatibility complex genes of human and cyprinid fish, finding greater pairwise differences between exons than between introns. Furthermore, distances determined from exons and from introns within the cyprinid sequences showed a positive correlation only in between-genus comparisons and not in within-genus comparisons. He concluded that recombination had separated the genealogical histories of introns and exons within these genes.
In reconstructing evolutionary history, we restricted consideration to synonymous or noncoding substitutions in the conservative evolution partition, the region remaining after exclusion of all sites that showed any indication of positive selection. We chose to base our inference of historical recombination on departures from proportionality of lengths of corresponding branches of genealogies of linked regions and differences in numbers of segregating sites (see Appendix). Other methods for detecting recombination (Posada 2002), including, for example, those based on comparisons of topology, have very little power for small samples. We found (1) significant nonproportionality of branch lengths between the genealogies of the S-RNase and 48A regions borne on the same haplotype (table 6) and (2) significant reductions in the number of segregating sites in 48A (table 7; see also table 4). Both aspects are consistent with historical recombination between 48A and S-RNase.
Nonproportionality of corresponding branch lengths might also reflect variation in substitution rate over evolutionary time. The rate of substitution of specificity-determining mutations is determined by the rate of origin of new S-alleles, effective population size, and the number of S-alleles segregating in the population (Vekemans and Slatkin 1994; Uyenoyama and Takebayashi 2003). Fluctuations in such aspects over the dozens of millions of years spanned by S-allele genealogies may well have contributed to variation in the rate of selectively driven substitutions. In contrast, the rate of neutral substitution is expected to depend only on the frequency at which neutral variants arise.
In table 6, boldface indicates all fixed-site comparisons that showed both significant differences in substitution rate and significant nonproportionality of corresponding branch lengths. In both the GSI and SSI systems studied, comparisons between the positive selection and conservative evolution partitions of the pistil gene indicated a higher substitution rate in the positive selection partition and nonproportionality of branch lengths. The positively selected partition showed an accelerated substitution rate even at the third codon position, suggesting the operation of positive selection at this position. However, nonproportionality in branch length became nonsignificant upon restriction of consideration to the third codon position, in accordance with neutral expectation.
Variation in the rate of selectively driven substitution is unlikely to account for the highly significant nonproportionality detected in a fixed-site comparison between the 3' UTR of 48A and the third codon position of the conservative evolution partition of S-RNase. In this case, we interpret the pattern as indicative of distinct genealogical histories of the 48A and S-RNase regions within the same haplotype.
Although comparisons between the pistil and pollen components of the Brassica SSI system also showed significant differences in overall substitution rate and nonproportional branch lengths, the pattern differed from that observed between S-RNase and 48A (tables 6 and 7). It is SP11, the determinant of pollen specificity, that showed the higher substitution rate. Also, because practically the entire coding region of SP11 showed evidence of positive or purifying selection, any inferences concerning its evolutionary history must be qualified by the possibility of strong selection across the entire gene. Actual variation in substitution rate across time periods and among sites may account for nonproportionality of branch lengths between partitions of the Brassica S-locus.
Genetic exchange within the pistil gene SRK may also have contributed to nonproportionality of branch lengths, in spite of our efforts to minimize this effect by excluding from our analysis sequences for which evidence of genetic exchange is strong (see Materials and Methods). The SRK gene was first isolated by virtue of its high sequence similarity to SLG, which was formerly regarded as the determinant of pistil specificity (Stein et al. 1991). Observations of patches showing much greater sequence similarity in paralogous comparisons between SRK and SLG within the same haplotype than in orthologous comparisons between different haplotypes provide evidence of some form of genetic exchange between the genes (Goring et al. 1993; Watanabe et al. 1994; Kusaba et al. 1997; Sato et al. 2002).
That genetic exchange within S-RNase (Wang et al. 2001) rather than between S-RNase and 48A has contributed to the nonproportionality of branch lengths in the S-RNase-based system of GSI remains a possibility. To the extent that genetic exchange within S-RNase might have served as a mechanism for the generation of new, positively selected S-specificities, it might also have contributed to the acceleration of substitution in S-RNase relative to 48A. However, a comparison of the third codon positions of the positive selection and conservative partitions of S-RNase indicated nonsignificant departures from proportionality of corresponding branch lengths (table 6). Furthermore, in combination with nonproportional branch lengths, 48A showed highly significant reductions in number of synonymous substitutions relative to S-RNase (table 7). One might consider attributing the difference in synonymous substitutions to acceleration in S-RNase rather than to reduction in 48A. Because neutral substitution depends on the rate of origin of neutral variants alone, and in particular because it is not influenced by linkage to selected sites (Birky and Walsh 1988), hitchhiking explanations would require the simultaneous creation of selected and neutral variants. Such coincident mutation might arise, for example, through genetic exchange of segments spanning both synonymous and nonsynonymous sites. Even so, the absence of detectable positive selection within 48A would appear to support our central conclusion that it may not correspond to pollen-S.
It is only comparisons involving the 3' UTR of 48A that indicated significant nonproportionality with regions within S-RNase borne on the same haplotype (table 6). This pattern raises the possibility that genetic transfer events involving 48A have been restricted to the 3' UTR. For example, proper control of the expression of 48A throughout the very long periods over which S-haplotypes have been maintained might have been achieved not through successive substitution of 48A alleles but rather through gene conversion targeted specifically to the 3' UTR. Even so, this explanation for nonproportionality of branch lengths between the 3' UTR of 48A and S-RNase would not account for the highly significant reduction in substitution rate relative to S-RNase evident in the coding region of 48A as well as the 3' UTR (table 7).
We suggest that incomplete linkage between 48A and S-RNase provides the most parsimonious interpretation of our results. This conclusion challenges the hypothesis that 48A serves as the primary determinant of pollen specificity in the solanaceous system of S-RNase-based GSI. Our estimate of a 15- to 20-fold reduction in genealogical depth in 48A provides information about the rate of recombination between 48A and S-RNase (cf. Strobeck 1980; Hudson and Kaplan 1988; Schierup, Charlesworth, and Vekemans 2000). We address this issue in a separate study.
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Neutral mutations arise within locus i (i = 0, 1) at rate vi per site per generation. Given the total time in the genealogy since the MRCA of the sampled genes, the number of substitutions within the genealogy follows a Poisson distribution with parameter
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Identical Substitution Rates at Absolutely Linked Loci
We first addressed whether the relative divergence at neutral sites within locus 0 and locus 1 is consistent with absolute linkage and identical rates of neutral substitution. We obtained per-locus estimates of the number of segregating sites (xi = nidi for di the estimated per-site distance) under an ML joint genealogy determined from sequences concatenated across loci. Absolute linkage entails a single total time with the genealogy for the two loci. Under the hypothesis {T0 = T1 = T, 0 =
1 =
}, equation A.1 reduces to
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The maximum likelihood estimate of T, obtained by maximizing the probability of observing the estimated distances under equation A.2, corresponds to
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To examine whether the observed distances are consistent with a common value of T for the loci, we determined the approximate probability of states showing deviations equal to or greater than observed. Designating locus 1 as the one that shows fewer substitutions than expected, the probability is
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Different Substitution Rates at Absolutely Linked Loci
For cases in which the hypothesis of a common total time and substitution rate across loci is rejected, we retained the estimate of xi obtained under the joint genealogy but estimated i for each locus separately. Under the hypothesis of a common total time but different substitution rates for the loci,
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Incomplete Linkage
An alternative interpretation of between-locus differences in the numbers of segregating sites is that the loci share a common rate of substitution but have different genealogical histories, perhaps reflecting ancestral recombination events. Accordingly, we estimated for each locus separately an independent genealogy and the number of segregating sites (xi). Under this model, estimates obtained from (equation A.6) have the interpretation
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Footnotes |
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