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The connections leading from genotype to fitness are not well understood, yet they are crucial for a diverse set of disciplines. Uncovering the general properties of biochemical pathways that influence ecologically important traits is an effective way to understand these connections. Enzyme flux control (or, control over pathway output) is one such pathway property. The flux-controlling enzyme in the antiherbivory aliphatic glucosinolate pathway of Arabidopsis thaliana has majority flux control under benign greenhouse conditions and has evidence of nonneutral evolution. However, it is unknown how patterns of flux control may change in different environments, or if insect herbivores respond to differences in pathway flux. We test this, first through genetic manipulation of the loci that code for the aliphatic glucosinolate pathway enzymes under a variety of environments (reduced water, reduced soil nutrients, leaf wounding and methyl jasmonate treatments), and find that flux control is consistently in the first enzyme of the pathway. We also find that a generalist herbivore, Trichoplusia ni, modifies its feeding behavior depending on the flux through the glucosinolate pathway. The influence over herbivore behavior combined with the consistency of flux control suggests that genes controlling flux might be repeatedly targeted by natural selection in diverse environments and species.

Strategies that enable species to persist in changing environments have historically been divided into ecological (distributional shifts and phenotypic plasticity) and evolutionary (adaptation and gene flow). However, most species will likely need to rely on a combination of approaches to mitigate

Recent studies have elucidated the ancient polyploid history of the Arabidopsis thaliana (Brassicaceae) genome. The studies concur that there was at least one polyploidy event occurring some 14.5 to 86 million years ago (Mya), possibly near the divergence of the Brassicaceae from its sister family, Cleomaceae. Using a comparative genomics approach, we asked whether this polyploidy event was unique to members of the Brassicaceae, shared with the Cleomaceae, or an independent polyploidy event in each lineage. We isolated and sequenced three genomic regions from diploid Cleome spinosa (Cleomaceae) that are each homoeologous to a duplicated region shared between At3 and At5, centered on the paralogs of SEPALLATA (SEP) and CONSTANS (CO). Phylogenetic reconstructions and analysis of synonymous substitution rates support the hypothesis that a genomic triplication in Cleome occurred independently of and more recently than the duplication event in the Brassicaceae. There is a strong correlation in the copy number (single versus duplicate) of individual genes, suggesting functionally consistent influences operating on gene copy number in these two independently evolving lineages. However, the amount of gene loss in Cleome is greater than in Arabidopsis. The genome of C. spinosa is only 1.9 times the size of A. thaliana, enabling comparative genome analysis of separate but related polyploidy events.

In multigene families, variation among loci and alleles can contribute to trait evolution. We explored patterns of functional and genetic variation in six duplicated Arabidopsis thaliana trypsin inhibitor (ATTI) loci. We demonstrate significant variation in constitutive and herbivore-induced transcription among ATTI loci that show, on average, 65% sequence divergence. Significant variation in ATTI expression was also found between two molecularly defined haplotype classes. Population genetic analyses for 17 accessions of A. thaliana showed that six ATTI loci arranged in tandem within 10 kb varied 10-fold in nucleotide diversity, from 0.0009 to 0.0110, and identified a minimum of six recombination events throughout the tandem array. We observed a significant peak in nucleotide and indel polymorphism spanning ATTI loci in the interior of the array, due primarily to divergence between the two haplotype classes. Significant deviation from the neutral equilibrium model for individual genes was interpreted within the context of intergene linkage disequilibrium and correlated patterns of functional differentiation. In contrast to the outcrosser Arabidopsis lyrata for which recombination is observed even within ATTI loci, our data suggest that response to selection was slowed in the inbreeding, annual A. thaliana because of interference among functionally divergent ATTI loci.

DNA sequence variation of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA from Arabis holboellii, A. drummondii, and its putative hybrid A. divaricarpa was analyzed to study hybrid speciation in a species system geographically covering nearly the entire North American continent. Based on molecular systematics the investigated species are better combined under the genus Boechera. Multiple intraindividual ITS copies were detected in numerous accessions of A. divaricarpa, and, to a minor extent, in the parental taxa. Comparative phylogenetic analysis demonstrates that reticulate evolution is common. Consequently, concerted evolution of ITS regions resulted in different types of ITS fragments not only in hybrid populations but also in one of the parental taxa, A. holboellii. Hybrid formation often occurred independently at different sites and at different times, which is reflected by ITS copies resampling the original parental sequence variation in different ways. Some biogeographic structuring of genetic diversity is apparent and mirrors postglacial migration routes. Hybridization, reticulation, and apomixis are assumed to be the major forces driving speciation processes in this species complex. Analysis of conserved regions and secondary structures of the ITS region provided no evidence that, in this system, hybrid ITS evolution is predominantly driven in a particular direction. However, two regions in the ITS1 and ITS2, respectively, show higher mutation rates than expected from outgroup comparisons. Strong evidence for the occurrence of apomixis in A. holboellii and A. divaricarpa has come from pollen size measurements and estimations of pollen quality, which favor the hypothesis that A. drummondii served as paternal hybridization partner more frequently than A. holboellii.

Glucosinolates are anionic thioglucosides that have become one of the most frequently studied groups of defensive metabolites in plants. When tissue damage occurs, the thioglucoside linkage is hydrolyzed by enzymes known as myrosinases, resulting in the formation of a variety of products that are active against herbivores and pathogens. In an effort to learn more about the molecular genetic and biochemical regulation of glucosinolate hydrolysis product formation, we analyzed leaf samples of 122 Arabidopsis ecotypes. A distinct polymorphism was observed with all ecotypes producing primarily isothiocyanates or primarily nitriles. The ecotypes Columbia (Col) and Landsberg erecta (Ler) differed in their hydrolysis products; therefore, the Col × Ler recombinant inbred lines were used for mapping the genes controlling this polymorphism. The major quantitative trait locus (QTL) affecting nitrile versus isothiocyanate formation was found very close to a gene encoding a homolog of a Brassica napus epithiospecifier protein (ESP), which causes the formation of epithionitriles instead of isothiocyanates during glucosinolate hydrolysis in the seeds of certain Brassicaceae. The heterologously expressed Arabidopsis ESP was able to convert glucosinolates both to epithionitriles and to simple nitriles in the presence of myrosinase, and thus it was more versatile than previously described ESPs. The role of ESP in plant defense is uncertain, because the generalist herbivore Trichoplusia ni (the cabbage looper) was found to feed more readily on nitrile-producing than on isothiocyanate-producing Arabidopsis. However, isothiocyanates are frequently used as recognition cues by specialist herbivores, and so the formation of nitriles instead of isothiocyanates may allow Arabidopsis to be less apparent to specialists.

Arabidopsis and other Brassicaceae produce an enormous diversity of aliphatic glucosinolates, a group of methionine (Met)-derived plant secondary compounds containing a β-thio-glucose moiety, a sulfonated oxime, and a variable side chain. We fine-scale mappedGSL-ELONG, a locus controlling variation in the side-chain length of aliphatic glucosinolates. Within this locus, a polymorphic gene was identified that determines whether Met is extended predominantly by either one or by two methylene groups to produce aliphatic glucosinolates with either three- or four-carbon side chains. Two allelic mutants deficient in four-carbon side-chain glucosinolates were shown to contain independent missense mutations within this gene. In cell-free enzyme assays, a heterologously expressed cDNA from this locus was capable of condensing 2-oxo-4-methylthiobutanoic acid with acetyl-coenzyme A, the initial reaction in Met chain elongation. The gene methylthioalkylmalate synthase1(MAM1) is a member of a gene family sharing approximately 60% amino acid sequence similarity with 2-isopropylmalate synthase, an enzyme of leucine biosynthesis that condenses 2-oxo-3-methylbutanoate with acetyl-coenzyme A.

DNA sequence variations of chalcone synthase (Chs) and Apetala3 gene promoters from 22 cruciferous plant species were analyzed to identify putative conserved regulatory elements. Our comparative approach confirmed the existence of numerous conserved sequences which may act as regulatory elements in both investigated promoters. To confirm the correct identification of a well-conserved UV-light-responsive promoter region, a subset of Chs promoter fragments were tested in Arabidopsis thaliana protoplasts. All promoters displayed similar light responsivenesses, indicating the general functional relevance of the conserved regulatory element. In addition to known regulatory elements, other highly conserved regions were detected which are likely to be of functional importance. Phylogenetic trees based on DNA sequences from both promoters (gene trees) were compared with the hypothesized phylogenetic relationships (species trees) of these taxa. The data derived from both promoter sequences were congruent with the phylogenies obtained from coding regions of other nuclear genes and from chloroplast DNA sequences. This indicates that promoter sequence evolution generally is reflective of species phylogeny. Our study also demonstrates the great value of comparative genomics and phylogenetics as a basis for functional analysis of promoter action and gene regulation.

Secondary metabolites are a diverse set of plant compounds believed to have numerous functions in plant-environment interactions. The large chemical diversity of secondary metabolites undoubtedly arises from an equally diverse set of enzymes responsible for their biosynthesis. However, little is known about the evolution of enzymes involved in secondary metabolism. We are studying the biosynthesis of glucosinolates, a large group of secondary metabolites, in Arabidopsis to investigate the evolution of enzymes involved in secondary metabolism. Arabidopsis contains natural variations in the presence of methylsulfinylalkyl, alkenyl, and hydroxyalkyl glucosinolates. In this article, we report the identification of genes encoding two 2-oxoglutarate--dependent dioxygenases that are responsible for this variation. These genes, AOP2 and AOP3, which map to the same position on chromosome IV, result from an apparent gene duplication and control the conversion of methylsulfinylalkyl glucosinolate to either the alkenyl or the hydroxyalkyl form. By heterologous expression in Escherichia and the correlation of gene expression patterns to the glucosinolate phenotype, we show that AOP2 catalyzes the conversion of methylsulfinylalkyl glucosinolates to alkenyl glucosinolates. Conversely, AOP3 directs the formation of hydroxyalkyl glucosinolates from methylsulfinylalkyl glucosinolates. No ecotype coexpressed both genes. Furthermore, the absence of functional AOP2 and AOP3 leads to the accumulation of the precursor methylsulfinylalkyl glucosinolates. A third member of this gene family, AOP1, is present in at least two forms and found in all ecotypes examined. However, its catalytic role is still uncertain.

We analyzed sequence variation for chalcone synthase (Chs) and alcohol dehydrogenase (Adh) loci in 28 species in the genera Arabidopsis and Arabis and related taxa from tribe Arabideae. Chs was single-copy in nearly all taxa examined, while Adh duplications were found in several species. Phylogenies constructed from both loci confirmed that the closest relatives of Arabidopsis thaliana include Arabidopsis lyrata, Arabidopsis petraea, and Arabidopsis halleri (formerly in the genus Cardaminopsis). Slightly more distant are the North American n = 7 Arabis (Boechera) species. The genus Arabis is polyphyletic-some unrelated species appear within this taxonomic classification, which has little phylogenetic meaning. Fossil pollen data were used to compute a synonymous substitution rate of 1.5 x 10 substitutions per site per year for both Chs and Adh. Arabidopsis thaliana diverged from its nearest relatives about 5 MYA, and from Brassica roughly 24 MYA. Independent molecular and fossil data from several sources all provide similar estimates of evolutionary timescale in the Brassicaceae.

Varied responses by specialist herbivores to glucosinolates could be a function of glucosinolate concentration or other correlated resistance factors, Herbivory by the specialist flea beetle Phyllotreta cruciferae (Goeze), and diamondback moth, Plutella xylostella (L.), varied curvilinearly with natural levels of glucosinolates in Brassica rapa ( syn.campestris ) (L.) such that maximum herbivory occurred at intermediate glucosinolate levels, Although the pattern was weak, decreases in herbivory at high concentrations of glucosinolates were enhanced by inoculating plants with the fungal pathogen Leptosphaeria maculans (Desm.) Ces. et de Not.(Ascomycetes), in laboratory experiments. This enhancement effect may have been caused by other correlated induced resistance factors produced by the plant or by resistance factors produced by the pathogen (independent of the host plant), Although specialist herbivores of mustards may have overcome glucosinolates, the apparent dose-dependent effect of glucosinolates suggest this herbivore counteradaptation may not have been complete, perhaps because of correlated resistance factors. Because a component of glucosinolate variation in B. rapa is heritable and because P. cruciferae can negatively affect plant fitness, the vried responses of specialist herbivores with glucosinolate concentrations reported here further suggest the possibility of disruptive selection on glucosinolates.

Studies of inbreeding depression in wild plants customarily compare the fitness of outcrossed progeny to progeny derived from one generation of self-pollination. We compare levels of inbreeding depression in a greenhouse in two populations of jewelweed using progeny derived from random outcrosses, one generation of self-pollination, and three generations of selling. The progeny have expected inbreeding coefficients of, respectively, 0, 0.5, and 0.875. Seedling survivorship declined linearly with the level of inbreeding in both populations. Inbreeding also increased the variability of emergence date. Maternal family membership affected early seedling performance and often interacted significantly with the level of inbreeding. In contrast, path analyses reveal that inbreeding had both negative linear and positive quadratic direct effects on seed and final plant weight, causing the highly inbred progeny to outperform progeny derived from one generation of selfing. These results suggest either the rapid purging of deleterious alleles or diminishing epistasis among the loci affecting these characters. It is not clear why the loci affecting survival responded differently.

Wade and Kalisz (1990) discuss approaches to identifying the causes of natural selection. They state that previous statistical analyses (e.g., Mitchell-Olds and Shaw, 1987) give "the impression that the quality of an analysis is equated with sophistication in multivariate statistics," and have "overshadowed the fact that the analysis of selection is essentially an ecological and not a statistical question." Wade and Kalisz recommend several procedures (experimental manipulation, explicit consideration of environmental effects, and understanding of ecological mechanisms), which are billed as an "empirical complement" to MitchellOlds and Shaw (1987). We have previously discussed interpretation and limitations of selection analyses (Mitchell-Olds and Shaw, 1987; all page references are from this paper). In addition to statistical issues, we advocated experimentation as an essential ingredient for biological interpretation. For example, we and many others have noted that correlation, even in a multivariate regression analysis, is not sufficient to imply causation ("Perhaps the most desirable approach to model validation is experimental manipulation to avoid the weaknesses inherent in a correlational approach," p. 1159). We also suggested "explicit consideration of environmental effects, . . . [which] permits detection of environment specific differences in the relationship of the characters to fitness" (p. I 1 5 5). Beyond experimental manipulation, we also have suggested that "functional analyses of the ecological importance of the characters may be used to support inferences from the regression approach" (p. 1 159). When results from correlational or regression analyses are in accord with known physiological or ecological mechanisms, then biological inferences are greatly strengthened. Biologists have long recognized limitations on biological interpretations based on correlational analyses, and the importance of experimentation for resolution of these questions. These points were emphasized in our 1987 paper, and have been discussed by experimentalists for many years (e.g., "causes and mechanisms must also be demonstrated with critical experiments," Endler, 1986, p. 201; additional references in Mitchell-Olds and Shaw, 1987). Wade and Kalisz present these ideas as their own original invention, with no reference to previous workers. Furthermore, their discussion mentions our 1987 paper, then appears to contrast their own views: "We [Wade and Kalisz] are concemed with the research program after this estimate of the fitness function is obtained. We suggest experimental manipulations.... We too are interested in the best unbiased estimate .... However, we treat even the best estimate of the selection gradient through multivariate analysis as an 'hypothesis'.... It can only suggest causation." We applaud Wade and Kalisz's suggestions for explicit consideration of environmental effects, experimental manipulation, and understanding of ecological (as well as physiological) mechanisms, having previously advocated this approach. Wade and Kalisz build on the previous work of many biologists, and suggest useful extensions to provide improved ecological understanding. Regression analysis is an important method for obtaining partial understanding of possible pattems of natural selection, and for suggesting hypotheses for future research. However, such correlational analyses are never sufficient for inference of causal pattems of natural selection. Attempts to understand causation are reinforced by experimental manipulations or examination of physiological or ecological mechanisms. "Our understanding of evolutionary processes will be greatly strengthened by intensive, long-term studies of wild populations employing a variety of techniques" (Mitchell-Olds and Shaw, 1987, p. 1160).

Recent theoretical work in quantitative genetics has fueled interest in measuring natural selection in the wild. We discuss statistical and biological issues that may arise in applications of Lande and Arnold's (1983) multiple-regression approach to measuring selection. We review assumptions involved in estimation and hypothesis testing in regression problems, and we note difficulties that frequently arise as a result of violation of these assumptions. In particular, multicollinearity (extreme intercorrelation of characters) and extrinsic, unmeasured factors affecting fitness may seriously complicate inference regarding selection. Further, violation of the assumption that residuals are normally distributed vitiates tests of significance. For this situation, we suggest applications of recently developed jackknife tests of significance. While fitness regression permits direct assessment of selection in a form suitable for predicting selection response, we suggest that the aim of inferring causal relationships about the effects of phenotypic characters on fitness is greatly facilitated by manipulative experiments. Finally, we discuss alternative definitions of stabilizing and disruptive selection.

Using field and greenhouse experiments, we tested two hypotheses that could account for the maintenance of outcrossing in Impatiens capensis. Seedlings derived from cleistogamous (CL) and chasmogamous (CH) flowers were grown under competitive conditions while flanked by neighbors that were either related or unrelated. In both experiments, CH progeny sometimes expressed more phenotypic variability than CL progeny. In the greenhouse experiment, CH progeny attained the same weight as CL progeny, and the relatedness of neighboring plants did not affect the growth of either type. In the field experiment, CL and CH progeny performed similarly when grown with related competitors. However, CH progeny were somewhat larger when planted with nonsibs, while CL progeny were somewhat smaller under those conditions. Thus, there is no evidence that either frequency-dependent selection or the avoidance of competition among siblings favors the maintenance of outcrossing in this species. We also modeled the relative variability of selfed and outcrossed progeny under several reproductive systems. When females mate with one male (progeny are full sibs), selfed progeny are often more variable than outcrossed progeny. When females engage in both selfing and outcrossing, variation among progeny is frequently maximized at an intermediate selfing rate. The sib-competition mechanism, under a range of genetic models, is not apt to promote outcrossing, since selfed progeny are commonly more variable than outcrossed progeny.

When variation in life-history characters is caused by many genes of small effect, then quantitative-genetic parameters may quantify constraints on rate and direction of microevolutionary change. I estimated heritabilities and genetic correlations for 16 life-history and morphological characters in two populations of Impatiens capensis, a partially self-pollinating herbaceous annual. The Madison population had little or no additive genetic variance for any of these characters, while the Milwaukee population had significant narrowsense heritabilities and genetic correlations for several traits, including adult size, which is highly correlated with fitness. All genetic correlations among fitness components were positive, hence there is no evidence for antagonistic pleiotropy among these traits. Dissimilarity of heritabilities in the two populations supports theoretical predictions that long-term changes in genetic variance-covariance patterns may occur when population sizes are small and selection is strong, as may occur in many plant species.