The evolution of quantitative traits in complex environments.

Species inhabit complex environments and respond to selection imposed by numerous abiotic and biotic conditions that vary in both space and time. Environmental heterogeneity strongly influences trait evolution and patterns of adaptive population differentiation. For example, heterogeneity can favor local adaptation, or can promote the evolution of plastic genotypes that alter their phenotypes based on the conditions they encounter. Different abiotic and biotic agents of selection can act synergistically to either accelerate or constrain trait evolution. The environmental context has profound effects on quantitative genetic parameters. For instance, heritabilities measured in controlled conditions often exceed those measured in the field; thus, laboratory experiments could overestimate the potential for a population to respond to selection. Nevertheless, most studies of the genetic basis of ecologically relevant traits are conducted in simplified laboratory environments, which do not reflect the complexity of nature. Here, we advocate for manipulative field experiments in the native ranges of plant species that differ in mating system, life-history strategy and growth form. Field studies are vital to evaluate the roles of disparate agents of selection, to elucidate the targets of selection and to develop a nuanced perspective on the evolution of quantitative traits. Quantitative genetics field studies will also shed light on the potential for natural populations to adapt to novel climates in highly fragmented landscapes. Drawing from our experience with the ecological model system Boechera (Brassicaceae), we discuss advancements possible through dedicated field studies, highlight future research directions and examine the challenges associated with field studies.

The calmodulin-binding transcription factor SIGNAL RESPONSIVE1 is a novel regulator of glucosinolate metabolism and herbivory tolerance in Arabidopsis.

The Arabidopsis Ca(2+)/calmodulin (CaM)-binding transcription factor SIGNAL RESPONSIVE1 (AtSR1/CAMTA3) was previously identified as a key negative regulator of plant immune responses. Here, we report a new role for AtSR1 as a critical component of plant defense against insect herbivory. Loss of AtSR1 function impairs tolerance to feeding by the generalist herbivore Trichoplusia ni as well as wound-induced jasmonate accumulation. The susceptibility of the atsr1 mutant is associated with decreased total glucosinolate (GS) levels. The two key herbivory deterrents, indol-3-ylmethyl (I3M) and 4-methylsulfinylbutyl (4MSOB), showed the most significant reductions in atsr1 plants. Further, changes in AtSR1 transcript levels led to altered expression of several genes involved in GS metabolism including IQD1, MYB51 and AtST5a. Overall, our results establish AtSR1 as an important component of plant resistance to insect herbivory as well as one of only three described proteins involved in Ca(2+)/CaM-dependent signaling to function in the regulation of GS metabolism, providing a novel avenue for future investigations of plant-insect interactions.

Ecological genomics of Boechera stricta: identification of a QTL controlling the allocation of methionine- vs branched-chain amino acid-derived glucosinolates and levels of insect herbivory.

In the Brassicaceae, glucosinolates influence the feeding, reproduction and development of many insect herbivores. Glucosinolate production and effects on herbivore feeding have been extensively studied in the model species, Arabidopsis thaliana and Brassica crops, both of which constitutively produce leaf glucosinolates mostly derived from the amino acid, methionine. Much less is known about the regulation or role in defense of glucosinolates derived from other aliphatic amino acids, such as the branched-chain amino acids (BCAA), valine and isoleucine. We have identified a glucosinolate polymorphism in Boechera stricta controlling the allocation to BCAA- vs methionine-derived glucosinolates in both leaves and seeds. B. stricta is a perennial species that grows in mostly undisturbed habitats of western North America. We have measured glucosinolate profiles and concentrations in 192 F(2) lines that have earlier been used for genetic map construction. We also performed herbivory assays on six F(3) replicates per F(2) line using the generalist lepidopteran, Trichoplusia ni. Quantitative trait locus (QTL) analysis identified a single locus controlling both glucosinolate profile and levels of herbivory, the branched chain-methionine allocation or BCMA QTL. We have delimited this QTL to a small genomic region with a 1.0 LOD confidence interval just 1.9 cm wide, which, in A. thaliana, contains approximately 100 genes. We also found that methionine-derived glucosinolates provided significantly greater defense than the BCAA-derived glucosinolates against feeding by this generalist insect herbivore. The future positional cloning of this locus will allow for testing various adaptive explanations.

Cis-regulatory evolution of chalcone-synthase expression in the genus Arabidopsis.

The contribution of cis-regulation to adaptive evolutionary change is believed to be essential, yet little is known about the evolutionary rules that govern regulatory sequences. Here, we characterize the short-term evolutionary dynamics of a cis-regulatory region within and among two closely related species, A. lyrata and A. halleri, and compare our findings to A. thaliana. We focused on the cis-regulatory region of chalcone synthase (CHS), a key enzyme involved in the synthesis of plant secondary metabolites. We observed patterns of nucleotide diversity that differ among species but do not depart from neutral expectations. Using intra- and interspecific F1 progeny, we have evaluated functional cis-regulatory variation in response to light and herbivory, environmental cues, which are known to induce CHS expression. We find that substantial cis-regulatory variation segregates within and among populations as well as between species, some of which results from interspecific genetic introgression. We further demonstrate that, in A. thaliana, CHS cis-regulation in response to herbivory is greater than in A. lyrata or A. halleri. Our work indicates that the evolutionary dynamics of a cis-regulatory region is characterized by pervasive functional variation, achieved mostly by modification of response modules to one but not all environmental cues. Our study did not detect the footprint of selection on this variation.

Plant-insect interactions.

Recent research shows partially overlapping signal transduction pathways controlling responses to wounding, insects, and pathogens. Chemical and behavioral assays show that plants release herbivore-specific volatiles, and that parasitic wasps can distinguish between these emission patterns. QTL mapping and candidate gene studies are beginning to identify polymorphic resistance genes, and ecological analyses provide information on the physiological and fitness costs of resistance. Such multidisciplinary approaches can elucidate the physiological causes and ecological consequences of plant-herbivore interactions.

Biogeographic distribution of polyploidy and B chromosomes in the apomictic Boechera holboellii complex.

The Boechera holboellii complex comprises B. holboellii and B. drummondii, both of which can reproduce through sex or apomixis. Sexuality is associated with diploid individuals, whereas apomictic individuals are diploid or triploid and may additionally have B chromosomes. Using flow cytometry and karyotype analysis, we have shown that B chromosomes (a) occur in both diploid and triploid apomictic B. holboellii, (b) may occur in triploid B. drummondii, and (c) are dispensable for the plant. Both diploid and triploid karyotypes are found in multiple chloroplast haplotypes of both species, suggesting that triploid forms have originated multiple times during the evolution of this complex. B chromosome carriers are found in geographically and genetically distinct populations, but it is unknown whether the extra chromosomes are shared by common descent (single origin) or have originated via introgressive hybridization and repeated transitions from diploidy to triploidy. Diploid plants containing the Bs reproduce apomictically, suggesting that the supernumerary elements are associated with apomixis. Finally, our analyses of pollen size and viability suggest that irregular chromosome segregation in some triploid lineages may lead to the generation of diploid individuals which carry the B chromosomes.

Interval mapping of viability loci causing heterosis in Arabidopsis.

The genetic basis of heterosis has implications for many problems in genetics and evolution. Heterosis and inbreeding depression affect human genetic diseases, maintenance of genetic variation, evolution of breeding systems, agricultural productivity, and conservation biology. Despite decades of theoretical and empirical studies, the genetic basis of heterosis has remained unclear. I mapped viability loci contributing to heterosis in Arabidopsis. An overdominant factor with large effects on viability mapped to a short interval on chromosome I. Homozygotes had 50% lower viability than heterozygotes in this chromosomal region. Statistical analysis of viability data in this cross indicates that observed viability heterosis is better explained by functional overdominance than by pseudo-overdominance. Overdominance sometimes may be an important cause of hybrid vigor, especially in habitually inbreeding species. Finally, I developed a maximum likelihood interval mapping procedure that can be used to examine chromosomal regions showing segregation distortion or viability selection.

Functional divergence in tandemly duplicated Arabidopsis thaliana trypsin inhibitor genes.

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.

The molecular basis of quantitative genetic variation in central and secondary metabolism in Arabidopsis.

To find the genes controlling quantitative variation, we need model systems where functional information on physiology, development, and gene regulation can guide evolutionary inferences. We mapped quantitative trait loci (QTLs) influencing quantitative levels of enzyme activity in primary and secondary metabolism in Arabidopsis. All 10 enzymes showed highly significant quantitative genetic variation. Strong positive genetic correlations were found among activity levels of 5 glycolytic enzymes, PGI, PGM, GPD, FBP, and G6P, suggesting that enzymes with closely related metabolic functions are coregulated. Significant QTLs were found influencing activity of most enzymes. Some enzyme activity QTLs mapped very close to known enzyme-encoding loci (e.g., hexokinase, PGI, and PGM). A hexokinase QTL is attributable to cis-acting regulatory variation at the AtHXK1 locus or a closely linked regulatory locus, rather than polypeptide sequence differences. We also found a QTL on chromosome IV that may be a joint regulator of GPD, PGI, and G6P activity. In addition, a QTL affecting PGM activity maps within 700 kb of the PGM-encoding locus. This QTL is predicted to alter starch biosynthesis by 3.4%, corresponding with theoretical models, suggesting that QTLs reflect pleiotropic effects of mutant alleles.

Statistical genetics of an annual plant, Impatiens capensis. II. Natural selection.

Measurement of natural selection on correlated characters provides valuable information on fitness surfaces, patterns of directional, stabilizing, or disruptive selection, mechanisms of fitness variation operating in nature, and possible spatial variation in selective pressures. We examined effects of seed weight, germination date, plant size, early growth, and late growth on individual fitness. Path analysis showed that most characters had direct or indirect effects on individual fitness, indicating directional selection. For most early life-cycle characters, indirect effects via later characters exceed the direct causal effect on fitness. Selection gradients were uniform across the experimental site. There was no evidence for stabilizing or disruptive selection. We discuss several definitions of stabilizing and disruptive selection. Although early events in the life of an individual have important causal effects on subsequent characters and fitness, there is no detectable genetic variance for most of these characters, so little or no genetic response to natural selection is expected.

Statistical genetics of an annual plant, Impatiens capensis. I. Genetic basis of quantitative variation.

Analysis of quantitative genetics in natural populations has been hindered by computational and methodological problems in statistical analysis. We developed and validated a jackknife procedure to test for existence of broad sense heritabilities and dominance or maternal effects influencing quantitative characters in Impatiens capensis. Early life cycle characters showed evidence of dominance and/or maternal effects, while later characters exhibited predominantly environmental variation. Monte Carlo simulations demonstrate that these jackknife tests of variance components are extremely robust to heterogeneous error variances. Statistical methods from human genetics provide evidence for either a major locus influencing germination date, or genes that affect phenotypic variability per se. We urge explicit consideration of statistical behavior of estimation and testing procedures for proper biological interpretation of statistical results.

Comparative quantitative trait loci mapping of aliphatic, indolic and benzylic glucosinolate production in Arabidopsis thaliana leaves and seeds.

Secondary metabolites are a diverse set of plant compounds believed to have numerous functions in plant-environment interactions. Despite this importance, little is known about the regulation of secondary metabolite accumulation. We are studying the regulation of glucosinolates, a large group of secondary metabolites, in Arabidopsis to investigate how secondary metabolism is controlled. We utilized Ler and Cvi, two ecotypes of Arabidopsis that have striking differences in both the types and amounts of glucosinolates that accumulate in the seeds and leaves. QTL analysis identified six loci determining total aliphatic glucosinolate accumulation, six loci controlling total indolic glucosinolate concentration, and three loci regulating benzylic glucosinolate levels. Our results show that two of the loci controlling total aliphatic glucosinolates map to biosynthetic loci that interact epistatically to regulate aliphatic glucosinolate accumulation. In addition to the six loci regulating total indolic glucosinolate concentration, mapping of QTL for the individual indolic glucosinolates identified five additional loci that were specific to subsets of the indolic glucosinolates. These data show that there are a large number of variable loci controlling glucosinolate accumulation in Arabidopsis thaliana.

Is the aneuploid chromosome in an apomictic Boechera holboellii a genuine B chromosome?

The Boechera holboellii complex comprises B. holboellii and B. drummondii, both of which can reproduce through sex or apomixis. Sexuality is associated with diploidy, whereas apomictic individuals can either be diploid, aneuploid or triploid. Aneuploid individuals are found in geographically and genetically distinct populations and contain a single extra chromosome. It is unknown whether the supernumerary chromosomes are shared by common descent (single origin) or have originated via introgressive hybridizations associated with the repeated transition from diploidy to triploidy. Diploid plants containing the extra chromosome(s) reproduce apomictically, suggesting that the supernumerary elements are associated with apomixis. In this study we compared flow cytometry data, chromosome morphology, and DNA sequences of sexual diploid and apomictic aneuploids in order to establish whether the extra chromosome fits the classical concept of a B chromosome. Karyotype analyses revealed that the supernumerary chromosome in the metaphase complement is heterochromatic and often smaller than the A chromosomes, and differs in length between apomictic plants from different populations. DNA sequence analyses furthermore demonstrated elevated levels of non-synonymous substitutions in one of the alleles, likely that on the aneuploid chromosome. Although the extra chromosome in apomictic Boechera does not go through normal reductional meiosis, in which it may get eliminated or accumulated by a B-chromosome-specific process, its variable size and heterochromatic nature does meet the remaining criteria for a genuine B chromosome in other species. Its prevalence and conserved genetic composition nonetheless implies that this chromosome, if truly a B, may be atypical with respect to its influence on its carriers.

Local adaptation across a climatic gradient despite small effective population size in the rare sapphire rockcress.

When assigning conservation priorities in endangered species, two common management strategies seek to protect remnant populations that (i) are the most genetically divergent or (ii) possess the highest diversity at neutral genetic markers. These two approaches assume that variation in molecular markers reflects variation in ecologically important traits and ignore the possibility of local adaptation among populations that show little divergence or variation at marker loci. Using common garden experiments, we demonstrate that populations of the rare endemic plant Arabis fecunda are physiologically adapted to the local microclimate. Local adaptation occurs despite (i) the absence of divergence at almost all marker loci and (ii) very small effective population sizes, as evidenced by extremely low levels of allozyme and DNA sequence polymorphism. Our results provide empirical evidence that setting conservation priorities based exclusively on molecular marker diversity may lead to the loss of locally adapted populations.

Genetics and evolution of insect resistance in Arabidopsis.

The genetic and molecular tools available in Arabidopsis allow identification of insect resistance genes. Many functional aspects of pest recognition and signal transduction are conserved in the defensive physiology of a broad range of plant species. Therefore, studies of insect resistance in Arabidopsis may be extended to functional genomics studies in many plant species of agricultural and ecological importance. Because of public concerns for field release of genetically modified organisms, naturally occurring genetic variation for resistance to insect herbivores will be valuable in plant breeding. Combined studies employing QTL mapping and candidate resistance genes are necessary to find and understand the genes responsible for variation in resistance. We review experiments showing that plant populations contain high levels of genetic variation for defensive physiology and disease and insect resistance, and that this variation can be manipulated to alter resistance and its components in a predictable fashion. In Arabidopsis, we can map the genes controlling physiological variation, and estimate the importance of regulatory or enzyme-encoding loci. Finally, we review functional genomics approaches for identification of insect resistance genes in Arabidopsis.

The molecular basis of quantitative genetic variation in natural populations.

DNA markers allow us to study quantitative trait loci (QTL) - the genes that control adaptation and quantitative variation. Experiments can map the genes responsible for quantitative variation and address the evolutionary and ecological significance of this variation. Recent studies suggest that major genes segregate within and among natural populations. It is now feasible to study the genes that cause morphological variation, life history trade-offs, heterosis and speciation. These methods can determine the role of epistasis and genotype-by-environment interaction in maintaining genetic variation. QTL mapping is an important tool used to address evolutionary and ecological questions of long-standing interest.

Population genetics of tandem trypsin inhibitor genes in Arabidopsis species with contrasting ecology and life history.

Duplicated genes are important in the evolution and ecology of plant-defences because herbivore and pathogen attack can be countered via functional diversification at two levels: among duplicated loci and within loci. We explore molecular sequence variation for two members of a defence-related gene family, Arabidopsis thaliana trypsin inhibitors (ATTI), in A. thaliana and a closely related species, A. lyrata subspp. petraea. A worldwide sample of the inbreeding annual A. thaliana had less genetic variation at two ATTI loci (piTOTAL

Evolution of plant resistance at the molecular level: ecological context of species interactions.

Molecular data regarding the diversity of plant loci involved in resistance to herbivores or pathogens are becoming increasingly available. These genes demonstrate variable patterns of diversity, suggesting that they differ in their evolutionary history. In parallel, the study of natural variation for resistance, generally conducted at the phenotypic level, has shown that resistance does not evolve solely under selection pressures exerted by enemies. Metapopulation dynamics and other ecological characteristics of interacting species also appear to have a large impact on resistance evolution. Until now, studies of resistance at the molecular level have been conducted separately from ecological studies in extant populations. Future progress requires an evolutionary approach integrating both molecular and ecological aspects of resistance evolution. Such an approach will contribute greatly to our understanding of the evolution of molecular diversity at loci involved in biotic stress.