Department of Ecology and Evolutionary Biology, University of California, Irvine
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Abstract |
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Introduction |
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There are two basic theoretical models that predict how defense genes evolve (Stahl et al. 1999
). One of these models predicts that variation in defense mechanisms is transitory because of positive selection imposed by parasites (Dawkins and Krebs 1979
). If this model accurately describes the evolution of plant defense genes, then these genes should contain low levels of genetic diversity, and population genetic analyses should reveal evidence of repeated positive selection. An alternative model predicts that variation in defense genes is maintained for long periods as a consequence of frequency-dependent selection associated with fluctuations in the frequencies of parasite genotypes (Jayakar 1970
; Clarke 1976
; May and Anderson 1983
; Seger 1988
; Frank 1992
) or costs associated with the expression of defense (Roy and Kirchner 2000
; Tiffin 2000)
. Under these models, defense genes should contain above-average levels of diversity, and this diversity should be spread between two, or possibly more, allelic classes whose most recent common ancestor is older than expected under a null model of neutral evolution.
Several molecular studies provide evidence that genes involved in pathogen recognition (i.e., R-genes) are subject to selection (Parniske et al. 1997
; Meyers et al. 1998
; Wang et al. 1998
). However, few studies have examined intraspecific diversity at individual resistance loci. Two investigations of intraspecific polymorphism of R-genes in Arabidopsis thaliana revealed patterns of polymorphisms that were consistent with selection having acted on these genes. One of these investigations detected strong evidence that the rpm1 gene had evolved in response to balancing selection, presumably resulting from fluctuations in selective pressure imposed by parasites (Stahl et al. 1999
). Results from the other study, which examined intraspecific polymorphism at the rps1 locus, were less clear but also suggestive of a nonneutral evolutionary past, perhaps also the result of some form of balancing selection (Caicedo, Schaal, and Kunkel 1999
).
Although these studies have begun to provide insight into the evolution of genes involved in defense against pathogens, they have focused primarily on just one aspect of defenseplant recognition of pathogens. Pathogen recognition can cause hypersensitive response and increased expression of a host of genes (i.e., induced defenses); however, R-genes themselves do not actually prevent or retard pathogen infection (Somssich and Hahlbrock 1998
). Few studies have examined the molecular evolution of the genes that code for proteins that actually limit the severity of parasite attack (but see Bishop, Dean, and Mitchell-Olds 2000)
.
Protease inhibitors (PIs) are among the best-studied plant defenses not encoded by R-genes (Garcia-Olmedo et al. 1987
; Ryan 1990
). These inhibitors are thought to be involved primarily in defense against herbivores, which rely on proteases to digest the proteins they consume (Ryan 1990
; Koiwa, Bressan, and Hasegawa 1997
). However, because pathogens rely on proteases to facilitate infection and spread within hosts, PIs may also limit the severity of pathogen infection (Ryan 1990
). Protease inhibitors may also be involved in the regulation of the plants' own proteases, especially in seeds where PIs can be found in high concentrations and may prevent the untimely degradation of seed storage proteins (Koiwa, Bressan, and Hasegawa 1997
). Several lines of empirical evidence support a role for protease inhibitors in plant defense: the expression of many PI genes is induced following mechanical or herbivore damage (Green and Ryan 1972
; Koiwa, Bressan, and Hasegawa 1997
), herbivores grow more slowly when reared on artificial diets containing PIs than when reared on artificial diets without PIs (Jongsma and Bolter 1997
), and transgenic plants expressing elevated levels of PIs incur less herbivore damage than control plants (Hilder et al. 1987
; Johnson et al. 1989
; McManus, White, and McGregor 1994
).
Protease inhibitors function as specific substrates for the proteases they inhibit. However, unlike normal substrates, which are cleaved by proteases and released quickly, the PI-protease complex is stable, and proteolysis of the inhibitor is limited and extremely slow (Laskowski 1985
; Garcia-Olmedo et al. 1987
). The specificity and efficacy of inhibition are determined by the degree of stereochemical complementation between the protease active site and a short inhibitory loop that extends out from the main body of the inhibitor molecule (Bode and Huber 1992
).
Molecular analyses of animal PIs have revealed some evidence for rapid evolution of these inhibitory loops. In particular, the reactive centers of homologous PIs isolated from related species are hypervariable (Hill et al. 1984
; Laskowski et al. 1987
; Creighton and Darby 1989
), and the reactive centers of duplicated PI genes diverge rapidly (Hill and Hastie 1987
). Although these observations have been interpreted as evidence for positive selection, presumably in response to selective pressure imposed by parasites (Hill and Hastie 1987
; Laskowski et al. 1987
; Creighton and Darby 1989
), little is known about the allelic diversity at specific PI loci.
In order to further our understanding of the molecular evolution and population genetics of plant protease inhibitors and plant defense mechanisms in general, we investigated the molecular diversity of the wip1 gene, a wound-induced serine protease inhibitor, in four taxa within the genus Zea. We also analyzed divergence of wip1 homologs isolated from species within the genera Zea, Tripsacum, Sorghum, and Oryza. Wip1 codes for a wound-induced protein with high similarity to members of the Bowman-Birk family of serine PIs (Eckelkamp, Ehmann, and Schopfer 1993
; Rohrmeier and Lehle 1993
). Like most Bowman-Birk PIs, wip1 is predicted to have two inhibitory domains (Rohrmeier and Lehle 1993
). However, unlike most Bowman-Birk PIs that inhibit both trypsin and chymotrypsin proteases (Ikenaka and Norioka 1986
), both inhibitory regions in wip1 are predicted to inhibit chymotrypsin proteases (Rohrmeier and Lehle 1993
). The specific objectives of this study were to determine (1) whether wip1 has a history of positive or balancing selection and (2) whether the evolutionary history of wip1 inhibitory loops differs from the evolutionary history of other parts of this gene.
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Materials and Methods |
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Sequence Analyses
Genealogies were constructed with PAUP* 4.0b (Swofford 1998
) using the neighbor-joining method (Saitou and Nei 1987
) with the HKY85 (Hasegawa, Kishino, and Yano 1985
) genetic distance. A sequence from T. dactyloides was used as an outgroup. Data were resampled 1,000 times for bootstrap analyses. All nucleotide sites were used for genealogical reconstruction. Estimates of genetic diversity,
(Tajima 1983
) and
(Watterson 1975
), were calculated separately on silent (synonymous and intron sites), synonymous, and nonsynonymous sites. Evidence for nonneutral evolution was investigated using the tests of Fu and Li (1993)
, Tajima (1989)
, Sawyer, Dyjhuizen, and Hartl (1987)
, and McDonald and Kreitman (1991)
(MK). All measures of polymorphism and tests of neutral evolution were calculated using DnaSP, version 3.5 (Rozas and Rozas 1999
).
Relative-rate tests between sequences from different genera were conducted using the method of Fitch (1976)
and Tajima (1993)
as implemented by MEGA (Kumar et al. 2000)
. Rate tests on Zea were conducted with the Z. mays ssp. mays 4b allele. This allele was chosen because it is located in the approximate middle of the wip1 genealogy and preliminary analyses showed that results obtained with this allele were typical of results obtained with other Zea alleles. Results from the rate tests were similar when either the O. sativa1 or the O. sativa2 sequence was used as the outgroup, and only results obtained with the O. sativa2 sequence are presented. All analyses involving O. sativa sequences were conducted on two alignments that differed in the location of an indel. Results from the tests differed little, and only results from the alignment that minimized the number of indels are presented.
Distribution of Changes in Different Gene Regions
A series of contingency tests were used to determine if polymorphic sites within Zea and fixed differences between Zea, T. dactyloides, S. bicolor, and O. sativa were distributed heterogeneously among four regions into which the wip1 sequence was divided a priori. Three of these four regions were predicted to have functional significance: two reactive-site loops active against chymotrypsin (designated chy1 and chy2) and a putative secretion signal sequence that is cleaved to form the mature protein (Rohrmeier and Lehle 1993
). The fourth region included all parts of the coding region that were not part of the above regions and is hereafter referred to as the structural region. The boundaries of the reactive-site loops were defined by the cysteine residues that are conserved across the Bowman-Birk family of inhibitors; these cysteines were included as part of the structural region of the molecule. The significance levels of the 4-by-2 contingency tests were evaluated using a
2 statistic, and the significance levels of the 2-by-2 tests were evaluated using Fisher's exact tests.
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Results |
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One caveat regarding these tests is that we do not know if these genes are duplicated, and thus we cannot be certain that tests were conducted on orthologs rather than paralogous gene copies. However, the pattern of divergence among sequences from the three taxa was what was expected for orthologous gene copies; i.e., the sequences from Zea and S. bicolor were more similar to one another than either was to either of the O. sativa sequences. Moreover, the genealogies of wip1-like genes had 100% bootstrap support for a branch containing O. sativa1 and O. sativa2 (see fig. 3 ), suggesting that the duplication event that resulted in two wip1-like sequences in O. sativa occurred after the divergence of Oryza from Zea, Tripsacum, and Sorghum. Taken together, these data suggest that we are making valid comparisons, and the relative-rate tests provide insight into the relative evolutionary rates of genes in Zea and S. bicolor.
The significance of the relative-rate tests was largely due to changes at replacement sites, providing further evidence for a role of selection in causing the significant rate heterogeneity in wip1. Synonymous-site changes were distributed on the Zea and S. bicolor branches with approximately equal frequencies (2 vs. 3 changes in the Zea and S. bicolor branches, respectively), whereas changes at replacement sites were heavily concentrated on the Zea branch (10 vs. 2 changes in the Zea and S. bicolor branches, respectively). Indels were also concentrated on the Zea branch (3 vs. 0 insertions in Zea and S. bicolor, respectively).
Distribution of Fixed and Polymorphic Sites in Different Gene Regions
Four-by-two contingency tests revealed no evidence (all P > 0.5) that polymorphic sites within Zea taxa are distributed heterogeneously among the four regions into which we a priori divided the wip1 gene. In contrast, the concentrations of both nucleic acid changes and indels that differentiated Zea, T. dactyloides, S. bicolor, and O. sativa differed significantly among the four regions (table 5
). Contingency tests conducted on synonymous and replacement sites separately revealed no evidence for significant heterogeneity in the distribution of synonymous sites (table 5
), but the distribution of replacement sites was highly heterogeneous (table 5
). Moreover, the ratio of replacement to synonymous substitutions was significantly heterogeneous, with the chy2 region having the highest ratio of Ka to Ks (table 5 ). A series of 2-by-2 contingency tests suggested that the significant heterogeneity detected by the 4-by-2 contingency tests was largely due to a greater frequency of evolutionary changes occurring in the chy2 region relative to the chy1 region and the structural regions (table 5
). All of the 2-by-2 contingency tests remained significant after a sequential Bonferroni correction for the six comparisons that were conducted within each class of changes. The 4-by-2 tests comparing the distributions of insertions and deletions and synonymous to replacement changes were not significant after a sequential Bonferroni correction for multiple tests (Rice 1989
); however, the probability that four of six tests were significant at P < 0.05 was very low.
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Discussion |
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Although significant rate heterogeneity may result from nonselective forces including changes in mutation rates, life history, or effective population sizes (Gillespie 1986
; Kreitman and Akashi 1995
), our evidence argues against this explanation for the significant rate heterogeneity in wip1. Unlike changes in selective pressure, these neutral evolutionary forces should affect entire genomes (Hudson, Kreitman, and Aguade 1987
). However, relative-rate tests conducted on eight other nuclear genes revealed no evidence for an elevated rate of evolution in Zea relative to S. bicolor. Moreover, the majority of changes that differentiate wip1 from Zea and S. bicolor are replacement changes that occurred during the evolution of Zea, as would be expected if selective forces were responsible for the significant rate heterogeneity. We also do not think that the elevated evolutionary rate detected in the Zea lineage is the result of a relaxation of selective constraint. A relaxation in selective constraint should be accompanied by high rates of intrataxon polymorphisms. However, polymorphisms in wip1 are within the range of polymorphisms found in neutrally evolving loci from Zea. Moreover, the chy2 region of wip1, which was largely responsible for the significant elevation in evolutionary rates, actually had lower levels of polymorphism than the structural regions of the gene. Thus, it seems more likely that the accelerated evolutionary rate in Zea is due to changes in selective forces. Rapid divergence of defense genes is consistent with some theoretical models that predict rapid divergence of defense alleles among related evolutionary lineages (Haldane 1949
; Clarke 1976
) and has been detected in chitinase, which is involved in plant defense against fungal pathogens, among Arabis species (Bishop, Dean, and Mitchell-Olds 2000)
.
In contrast to the results from these intergeneric analyses, results from intraspecific analyses do not indicate that wip1 has evolved in response to selection. The intraspecific and intergeneric tests may produce inconsistent results because the intraspecific tests for selection may have low statistical power (Wayne and Simonsen 1998
). We propose two possible biological reasons for the inconsistency. One of these possibilities is that there have been temporal fluctuations in selective pressures that have acted on wip1. In particular, if wip1 does evolve in response to selection imposed by herbivores and pathogens, then bouts of selection resulting from population outbreaks or shifts in the communities of herbivores and pathogens that attack Zea may have caused rapid evolutionary change. However, if the most recent bout of selection has been followed by a long period of selective neutrality, the intraspecific analyses will not detect evidence of selection, i.e., reduced diversity, because the signal has been lost through the accumulation of genetic diversity.
An alternative biological explanation for no evidence of selection being detected with the intraspecific tests is that wip1 evolves in a manner that is not detected by these analyses (Bergelson et al. 2001)
. For example, when a new defense allele enters a population, there may be no effective counterdefenses in the parasite population, and thus that allele may confer a selective advantage and increase in frequency. However, as that new defense allele increases in frequency, parasite counterdefenses will be selectively favored, and by the time the new defense allele becomes common, parasite counterdefenses may also be common, negating the selective advantage that was initially associated with the defense allele. Once counterdefenses have evolved, the once-advantageous defense allele may be selectively neutral and gradually lost from a population. In other words, wip1 alleles may be selectively favored only when they are uncommon and have been recently introduced into a population; i.e., wip1 alleles experience novel allele advantage. Because novel allele advantage would not result in either fixation of new alleles or the maintenance of alleles via balancing selection, results from molecular tests of nonneutral evolution, which are generally designed to detect either positive or balancing selection, may be consistent with a neutral evolutionary history, even though selection is acting.
Evolutionary Rates Differ Between the Two Inhibitory Loops
In addition to finding that wip1 has evolved faster in Zea than either S. bicolor or T. dactyloides, we found evidence that the two inhibitory loops of wip1 have diverged at significantly different rates. Among 50 sequences from the four genera, including the duplicated genes in O. sativa, we detected only a single, synonymous, change in the chy1 inhibitory loop. The lack of divergence in the chy1 inhibitory loop is surprising given that the majority of previous investigations of PIs have revealed evidence of elevated rates of evolutionary change and hypervariability of amino acids in inhibitory loops (Hill and Hastie 1987
; Laskowski et al. 1987
; Creighton and Darby 1989
; but see Beuning, Spriggs, and Christeller 1994
). Similarly, the duplicated genes in O. sativa do not exhibit high rates of divergence in either of the inhibitory loops, even though these two genes differ at more than 36 sites, indicating that sufficient time has passed for differences to accumulate (data not shown). Taken together, these results show that rapid evolutionary divergence of inhibitor loops may not be as general a phenomenon among plant protease inhibitors as previously thought (Creighton and Darby 1989
).
In contrast to the conserved chy1 region, the chy2 inhibitory loop has diverged rapidly. Although we cannot reject the possibility that chy2 has evolved neutrally, several aspects of the data are suggestive of a nonneutral evolutionary history. In particular, relative to other gene regions and other nuclear genes, the chy2 inhibitory loop has a higher ratio of nonconservative to conservative amino acid changes (data not shown), a higher ratio of fixed differences to polymorphic sites, an excess of fixed indels, and a significantly higher rate of divergence among Zea, Sorghum, Tripsacum, and Oryza, particularly at nonsynonymous sites (table 5 ). Many of these differences are not significantly different from expectations under a neutral model but are significantly different in the chy1 and chy2 regions (table 5 ). However, given that the chy2 region contains only 15 amino acids within Zea, and fewer than that in the other taxa, there was very little power to actually detect significant differences. In summary, the evidence for positive selection having acted on the chy2 region is equivocal, but the evidence for chy1 and chy2 regions having different selective histories is strong. An explanation for why these two inhibitory regions, which are predicted to have very different biochemical functions (Rohermeier and Lehel 1993), exhibit such different patterns of polymorphism and divergence will require more detailed analyses of these regions' biochemical and ecological functions.
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Acknowledgements |
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Footnotes |
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1 Present address: Department of Plant Biology, University of Minnesota, St. Paul.
2 Keywords: plant defense
induced resistance
coevolution
herbivory
maize
relative-rate tests
3 Address for correspondence and reprints: Peter Tiffin, Department of Plant Biology, University of Minnesota, 220 Biological Science Center, 1445 Gortner Avenue, St. Paul, Minnesota 51088. ptiffin{at}uci.edu
.
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