Characterization of the plant-specific BREVIS RADIX gene family reveals limited genetic redundancy despite high sequence conservation

To date, the function of most genes in the Arabidopsis (Arabidopsis thaliana) genome is unknown. Here we present the first analysis of the novel, plant-specific BRX (BREVIS RADIX) gene family. BRX has been identified as a modulator of root growth through a naturally occurring loss-of-function allele. The biochemical function of BRX is enigmatic, however several domains in BRX are conserved in the proteins encoded by the related BRX-like (BRXL) genes. The similarity between Arabidopsis BRXL proteins within these domains ranges from 84% to 93%. Nevertheless, analysis of brx brx-like multiple mutants indicates that functional redundancy of BRXLs is limited. This results mainly from differences in protein activity, as demonstrated by assaying the propensity of constitutively expressed BRXL cDNAs to rescue the brx phenotype. Among the genes tested, only BRXL1 can replace BRX in this assay. Nevertheless, BRXL1 does not act redundantly with BRX in vivo, presumably because it is expressed at a much lower level than BRX. BRX and BRXL1 similarity is most pronounced in a characteristic tandem repeat domain, which we named BRX domain. One copy of this domain is also present in the PRAF (PH, RCC1, and FYVE)-like family proteins. The BRX domain mediates homotypic and heterotypic interactions within and between the BRX and PRAF protein families in yeast (Saccharomyces cerevisiae), and therefore likely represents a novel protein-protein interaction domain. The importance of this domain for BRX activity in planta is underscored by our finding that expression of the C-terminal fragment of BRX, comprising the two BRX domains, is largely sufficient to rescue the brx phenotype.     

Soil microbes alter plant fitness under competition and drought

Plants exist across varying biotic and abiotic environments, including variation in the composition of soil microbial communities. The ecological effects of soil microbes on plant communities are well known, whereas less is known about their importance for plant evolutionary processes. In particular, the net effects of soil microbes on plant fitness may vary across environmental contexts and among plant genotypes, setting the stage for microbially mediated plant evolution. Here, we assess the effects of soil microbes on plant fitness and natural selection on flowering time in different environments. We performed two experiments in which we grew Arabidopsis thaliana genotypes replicated in either live or sterilized soil microbial treatments, and across varying levels of either competition (isolation, intraspecific competition or interspecific competition) or watering (well‐watered or drought). We found large effects of competition and watering on plant fitness as well as the expression and natural selection of flowering time. Soil microbes increased average plant fitness under interspecific competition and drought and shaped the response of individual plant genotypes to drought. Finally, plant tolerance to either competition or drought was uncorrelated between soil microbial treatments suggesting that the plant traits favoured under environmental stress may depend on the presence of soil microbes. In summary, our experiments demonstrate that soil microbes can have large effects on plant fitness, which depend on both the environment and individual plant genotype. Future work in natural systems is needed for a complete understanding of the evolutionary importance of interactions between plants and soil microorganisms.     

Heterogeneous selection at specific loci in natural environments in Arabidopsis thaliana

Genetic variation for quantitative traits is often greater than that expected to be maintained by mutation in the face of purifying natural selection. One possible explanation for this observed variation is the action of heterogeneous natural selection in the wild. Here we report that selection on quantitative trait loci (QTL) for fitness traits in the model plant species Arabidopsis thaliana differs among natural ecological settings and genetic backgrounds. At one QTL, the allele that enhanced the viability of fall-germinating seedlings in North Carolina reduced the fecundity of spring-germinating seedlings in Rhode Island. Several other QTL experienced strong directional selection, but only in one site and seasonal cohort. Thus, different loci were exposed to selection in different natural environments. Selection on allelic variation also depended upon the genetic background. The allelic fitness effects of two QTL reversed direction depending on the genotype at the other locus. Moreover, alternative alleles at each of these loci caused reversals in the allelic fitness effects of a QTL closely linked to TFL1, a candidate developmental gene displaying nucleotide sequence polymorphism consistent with balancing selection. Thus, both environmental heterogeneity and epistatic selection may maintain genetic variation for fitness in wild plant species.     

The genetics of chutes and ladders: a community genetics approach to tritrophic interactions

Community genetics research has firmly established that intraspecific genetic variation in single populations can have large extended ecological consequences for populations and entire communities of organisms. Here, we sought to understand the bottom‐up effects of plant genetic variation on herbivore preference and performance, and the top–down control of predators on herbivores and their joint effects on plant fitness and evolution. Following three ecological genetics field experiments we detected heritable variation in plant traits that influenced both the preference and performance of a specialist weevil on Oenothera biennis. However, the weevil's preference and performance were not genetically correlated among O. biennis plant genotypes. Although predators and parasitoids were abundant, predators had no detectable effect on weevil performance because high egg and larval mortality was caused by non‐predatory factors such as intraspecific competition. Finally, neither the specialist weevil nor predators influenced plant fitness. Our results suggest that the focal tritrophic community studied here is primarily shaped by the bottom–up effects of plant genetic variation on herbivores, while top–down effects have no clear impacts on O. biennis fitness or evolution. We suggest that future studies should incorporate plant intraspecific genetic variation as a fundamental part of tritrophic interactions including their eco‐evolutionary dynamics.     

The maintenance of heritable variation through social competition

The paradoxical persistence of heritable variation for fitness-related traits is an evolutionary conundrum that remains a preeminent problem in evolutionary biology. Here we describe a simple mechanism in which social competition results in the evolutionary maintenance of heritable variation for fitness related traits. We demonstrate this mechanism using a genetic model with two primary assumptions: the expression of a trait depends upon success in social competition for limited resources; and competitive success of a genotype depends on the genotypes that it competes against. We find that such social competition generates heritable (additive) genetic variation for "competition-dependent" traits. This heritable variation is not eroded by continuous directional selection because, rather than leading to fixation of favored alleles, selection leads instead to allele frequency cycling due to the concerted coevolution of the social environment with the effects of alleles. Our results provide a mechanism for the maintenance of heritable variation in natural populations and suggest an area for research into the importance of competition in the genetic architecture of fitness related traits.     

FRIGIDA-independent variation in flowering time of natural Arabidopsis thaliana accessions

FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) are two genes that, unless plants are vernalized, greatly delay flowering time in Arabidopsis thaliana. Natural loss-of-function mutations in FRI cause the early flowering growth habits of many A. thaliana accessions. To quantify the variation among wild accessions due to FRI, and to identify additional genetic loci in wild accessions that influence flowering time, we surveyed the flowering times of 145 accessions in long-day photoperiods, with and without a 30-day vernalization treatment, and genotyped them for two common natural lesions in FRI. FRI is disrupted in at least 84 of the accessions, accounting for only approximately 40% of the flowering-time variation in long days. During efforts to dissect the causes for variation that are independent of known dysfunctional FRI alleles, we found new loss-of-function alleles in FLC, as well as late-flowering alleles that do not map to FRI or FLC. An FLC nonsense mutation was found in the early flowering Van-0 accession, which has otherwise functional FRI. In contrast, Lz-0 flowers late because of high levels of FLC expression, even though it has a deletion in FRI. Finally, eXtreme array mapping identified genomic regions linked to the vernalization-independent, late-flowering habit of Bur-0, which has an alternatively spliced FLC allele that behaves as a null allele.     

Density dependent selection incorporating intraspecific competition 1. A haploid model

A haploid model is introduced and analyzed in which intraspecific competition is incorporated within a density dependent framework. It is assumed that each genotype has a unique carrying capacity corresponding to the equilibrium population size when fixed for that type. Each genotypic fitness at a single multi-allelic locus is a function of a distinctive effective population size formed by adding the numbers of each genotype present, weighted by an intraspecific competition coefficient. As a result, the fitnesses depend upon the relative frequencies of the various genotypes as well as the total population size. Intergenotypic interactions can have a profound effect upon the outcome of the population. In particular, when the density effect of one individual upon another depends upon their respective genotypes, a unique stable interior equilibrium is possible in which all alleles are present. This stands in contrast to the purely density dependent haploid system in which the only possible stable state corresponds to fixation for the type with the highest carrying capacity. In the present model selective advantage is determined by a balance between carrying capacity and sensitivity to density pressures from other genotypes. Fixation for the genotype with the highest carrying capacity, for instance, will not be stable if it exerts a sufficiently weak competitive effect upon the other genotypes. In the diallelic case, maintenance of both alleles at a stable equilibrium requires that the net intragenotypic competition between individuals of like genotype be stronger than that between unlike types. As for purely density regulated systems, there may be no stable equilibria and/or regular and chaotic cycling may occur. The results may also be interpreted in terms of a discrete time model of interspecific competition with each haplotype representing a different species.     

Estimates of natural selection due to protein tertiary structure inform the ancestry of biallelic loci

We consider the inference of which of two alleles is ancestral when the alleles have a single nonsynonymous difference and when natural selection acts via protein tertiary structure. Whereas the probability that an allele is ancestral under neutrality is equal to its frequency, under selection this probability depends on allele frequency and on the magnitude and direction of selection pressure. Although allele frequencies can be well estimated from intraspecific data, small fitness differences have a large evolutionary impact but can be difficult to estimate with only intraspecific data. Methods for predicting aspects of phenotype from genotype can supplement intraspecific sequence data. Recently developed statistical techniques can assess effects of phenotypes, such as protein tertiary structure on molecular evolution. While these techniques were initially designed for comparing protein-coding genes from different species, the resulting interspecific inferences can be assigned population genetic interpretations to assess the effect of selection pressure, and we use them here along with intraspecific allele frequency data to estimate the probability that an allele is ancestral. We focus on 140 nonsynonymous single nucleotide polymorphisms of humans that are in proteins with known tertiary structures. We find that our technique for employing protein tertiary structure information yields some biologically plausible results but that it does not substantially improve the inference of ancestral human allele types.     

Genotype and maternal environment affect belowground interactions between Arabidopsis thaliana and its competitors

Ecological interactions between different species are not fixed, but they may depend, at least to some extent, on the particular genotypes involved as well as on the environmental conditions experienced by previous generations. We used a set of natural genotypes of Arabidopsis thaliana , that previously experienced contrasting nutrient and herbivory conditions, to test for the influences of genetic variation and maternal effects on competitive interactions between Arabidopsis and the weedy annuals Anagallis arvensis and Senecio vulgaris . We used activated carbon to discriminate between resource competition and allelopathy components of plant-plant interactions. There was a clear competitive hierarchy: Senecio > Arabidopsis > Anagallis . Although we found no evidence for allelopathic potential of Arabidopsis , our results indicate that both Anagallis and Senecio exerted negative (direct or indirect) allelopathic effects on Arabidopsis . There were significant differences among Arabidopsis genotypes in their competitive effects on both neighbor species, as well as in their response to competition. Maternal environments significantly influenced not only the growth and fitness of Arabidopsis itself, but also its competitive effect on Anagallis . We found, however, no evidence that maternal environments affected the competitive effect on Senecio or overall competitive response of Arabidopsis . Generally, resource competition played a greater role than allelopathy, and genotype effects were more important than maternal effects. Our study demonstrates that ecological interactions, such as plant competition, are complex and multi-layered, and that, in particular, the influence of genetic variation on interactions with other species should not be overlooked.     

A trade-off in stand size effects in the reproductive biology of a declining tropical conifer Callitris intratropica

Because wind pollination is inefficient over longer distances, plants dependent on it may suffer Allee effects (lower individual reproductive fitness with lower density). However, at higher density, individual reproductive fitness may suffer because of intraspecific competition. We investigate density-dependent effects, via stand size, on cone and seed production and seed germinability in a conifer endemic to tropical Australia. Callitris intratropica R.T. Baker & H.G. Smith is an obligate-seeding tree that often occurs in monodominant stands embedded within savannas and on the fringes of monsoon forests. We found that isolated trees (50–300 m from stands) were taller, of broader profile, and produced approximately twice the number of cones (~407 cones per tree) as those in large stands (~173 cones per tree), suggesting that monodominance generates intraspecific competition. The number of seeds per cone (27 seeds) was not related to stand size. However, a contrasting effect in which seed germinability was higher in large stands (~20 vs. <10 % in small stands) was approximately compensatory and consistent with an Allee effect of wind pollination. The net effect of an approximately even trade-off between cone production and seed germinability was that there was neither an Allee or density-dependent effect of stand size on fitness, measured as the number of germinable seeds per tree. Nevertheless, because the likelihood of cross-fertilisation declines with distance, the ability of C. intratropica to persist as very isolated individuals may be limited.     

Natural genetic variation in Arabidopsis: tools, traits and prospects for evolutionary ecology

BACKGROUND: The model plant Arabidopsis thaliana (Arabidopsis) shows a wide range of genetic and trait variation among wild accessions. Because of its unparalleled biological and genomic resources, the potential of Arabidopsis for molecular genetic analysis of this natural variation has increased dramatically in recent years. SCOPE: Advanced genomics has accelerated molecular phylogenetic analysis and gene identification by quantitative trait loci (QTL) mapping and/or association mapping in Arabidopsis. In particular, QTL mapping utilizing natural accessions is now becoming a major strategy of gene isolation, offering an alternative to artificial mutant lines. Furthermore, the genomic information is used by researchers to uncover the signature of natural selection acting on the genes that contribute to phenotypic variation. The evolutionary significance of such genes has been evaluated in traits such as disease resistance and flowering time. However, although molecular hallmarks of selection have been found for the genes in question, a corresponding ecological scenario of adaptive evolution has been difficult to prove. Ecological strategies, including reciprocal transplant experiments and competition experiments, and utilizing near-isogenic lines of alleles of interest will be a powerful tool to measure the relative fitness of phenotypic and/or allelic variants. CONCLUSIONS: As the plant model organism, Arabidopsis provides a wealth of molecular background information for evolutionary genetics. Because genetic diversity between and within Arabidopsis populations is much higher than anticipated, combining this background information with ecological approaches might well establish Arabidopsis as a model organism for plant evolutionary ecology.     

Ecological interactions and the fitness effect of water‐use efficiency: Competition and drought alter the impact of natural MPK12 alleles in Arabidopsis

The presence of substantial genetic variation for water‐use efficiency (WUE) suggests that natural selection plays a role in maintaining alleles that affect WUE. Soil water deficit can reduce plant survival, and is likely to impose selection to increase WUE, whereas competition for resources may select for decreased WUE to ensure water acquisition. We tested the fitness consequences of natural allelic variation in a single gene (MPK12) that influences WUE in Arabidopsis, using transgenic lines contrasting in MPK12 alleles, under four treatments; drought/competition, drought/no competition, well‐watered/competition, well‐watered/no competition. Results revealed an allele × environment interaction: Low WUE plants performed better in competition, resulting from increased resource consumption. Contrastingly, high WUE individuals performed better in no competition, irrespective of water availability, presumably from enhanced water conservation and nitrogen acquisition. Our findings suggest that selection can influence MPK12 evolution, and represents the first assessment of plant fitness resulting from natural allelic variation at a single locus affecting WUE.     

Intraspecific competition facilitates the evolution of tolerance to insect damage in the perennial plant Solanum carolinense

Tolerance to herbivory (the degree to which plants maintain fitness after damage) is a key component of plant defense, so understanding how natural selection and evolutionary constraints act on tolerance traits is important to general theories of plant–herbivore interactions. These factors may be affected by plant competition, which often interacts with damage to influence trait expression and fitness. However, few studies have manipulated competitor density to examine the evolutionary effects of competition on tolerance. In this study, we tested whether intraspecific competition affects four aspects of the evolution of tolerance to herbivory in the perennial plant Solanum carolinense: phenotypic expression, expression of genetic variation, the adaptive value of tolerance, and costs of tolerance. We manipulated insect damage and intraspecific competition for clonal lines of S. carolinense in a greenhouse experiment, and measured tolerance in terms of sexual and asexual fitness components. Compared to plants growing at low density, plants growing at high density had greater expression of and genetic variation in tolerance, and experienced greater fitness benefits from tolerance when damaged. Tolerance was not costly for plants growing at either density, and only plants growing at low density benefited from tolerance when undamaged, perhaps due to greater intrinsic growth rates of more tolerant genotypes. These results suggest that competition is likely to facilitate the evolution of tolerance in S. carolinense, and perhaps in other plants that regularly experience competition, while spatio-temporal variation in density may maintain genetic variation in tolerance.     

A novel cost of R gene resistance in the presence of disease

Resistance responses can impose fitness costs when pests are absent. Here, we test whether the induction of resistance can decrease fitness even in plants under attack; we call this potential outcome a net cost with attack. Using lines in which genetic background was controlled, we investigated whether susceptible Arabidopsis thaliana plants can outperform R gene resistant plants when infected with pathogens. For the R gene RPS2, there was a fitness benefit of resistance in the presence of intraspecific competition, but there was a net cost in the absence of competition: resistant plants produced less seed than susceptible plants even though infected with Pseudomonas syringae. This net cost was primarily due to overcompensation by susceptible plants, which occurred because of a developmental response to infection. For the R gene RPP5, there was no fitness effect of resistance without competition but a net cost when plants were infected with Peronospora parasitica in the presence of competition. This net cost was due to a reduction in the fitness of infected, resistant plants and complete compensation in susceptible plants. A spatially variable model suggests that a trade-off between net benefits and net costs with attack may help explain the persistence of individuals lacking R gene resistance to disease.     

Trade-offs between male and female reproduction associated with allozyme variation in phosphoglucoisomerase in an annual plant (Clarkia unguiculata: Onagraceae)

The genotype of an individual for allozymes such as phosphoglucoisomerase (Pgi) is often not neutral with regard to fitness. Studies of several taxa have found consistent fitness differences among Pgi genotypes expressing different allozymes. We conducted a greenhouse experiment with Clarkia unguiculata to determine whether allelic variation at the Pgi-C1 locus may affect components of male and female function. We found significant differences in siring success between pollen donors homozygous for different Pgi alleles. When a mixture of pollen was applied to stigmas under conditions of gametophytic competition (more pollen deposited on stigmas than there are ovules available to fertilize), donors homozygous for the C allele of Pgi sired more seeds per fruit than B-allele donors. Differences between genotypes with respect to female fertility per fruit contrasted with the male advantage associated with the C allele. Recipients homozygous for the C allele produced fruits with more aborted seeds and fewer viable seeds than recipients homozygous for the B allele. These results suggest that allelic variation at a single locus may have opposing effects on male and female reproductive success in C. unguiculata, and that trade-offs between the two types of reproductive success could contribute to the maintenance of variation at the Pgi-C1 locus.     

Density-Dependent Selection Incorporating Intraspecific Competition. II. a Diploid Model

A diploid model is introduced and analyzed in which intraspecific competition is incorporated within the context of density-regulated selection. It is assumed that each genotype has a unique carrying capacity corresponding to the equilibrium population size when only that type is present. Each genotypic fitness at a single diallelic autosomal locus is a decreasing function of a distinctive effective population size perceived as a result of intraspecific competition. The resulting fitnesses are both density and frequency dependent with selective advantage determined by a balance between genotypic carrying capacity and sensitivity to intraspecific competition. A major finding is that intergenotypic interactions may allow genetic variation to be more easily maintained than in the corresponding model of purely density-dependent selection. In addition, numerical study confirms the possible existence of multiple interior equilibria and that neither overdominance in fitness nor carrying capacity is necessary for stability. The magnitude of the equilibrium population size and optimization principles are also discussed.     

Analysis of the molecular basis of flowering time variation in Arabidopsis accessions

Allelic variation at the FRI (FRIGIDA) and FLC (FLOWERING LOCUS C) loci are major determinants of flowering time in Arabidopsis accessions. Dominant alleles of FRI confer a vernalization requirement causing plants to overwinter vegetatively. Many early flowering accessions carry loss-of-function fri alleles containing one of two deletions. However, some accessions categorized as early flowering types do not carry these deletion alleles. We have analyzed the molecular basis of earliness in five of these accessions: Cvi, Shakhdara, Wil-2, Kondara, and Kz-9. The Cvi FRI allele carries a number of nucleotide differences, one of which causes an in-frame stop codon in the first exon. The other four accessions contain nucleotide differences that only result in amino acid substitutions. Preliminary genetic analysis was consistent with Cvi carrying a nonfunctional FRI allele; Wil-2 carrying either a defective FRI or a dominant suppressor of FRI function; and Shakhdara, Kondara, and Kz-9 carrying a functional FRI allele with earliness being caused by allelic variation at other loci including FLC. Allelic variation at FLC was also investigated in a range of accessions. A novel nonautonomous Mutator-like transposon was found in the weak FLC allele in Landsberg erecta, positioned in the first intron, a region required for normal FLC regulation. This transposon was not present in FLC alleles of most other accessions including Shakhdara, Kondara, or Kz-9. Thus, variation in Arabidopsis flowering time has arisen through the generation of nonfunctional or weak FRI and FLC alleles.     

Heterosis in hybrids within and between yeast species

The performance of hybrids relative to their parents is an important factor in speciation research. We measured the growth of 46 Saccharomyces yeast F1 interspecific and intraspecific hybrids, relative to the growth of each of their parents, in pairwise competition assays. We found that the growth of a hybrid relative to the average of its parents, a measure of mid‐parent heterosis, correlated with the difference in parental growth relative to their hybrid, a measure of phenotypic divergence, which is consistent with simple complementation of low fitness alleles in one parent by high fitness alleles in the other. Interspecific hybrids showed stronger heterosis than intraspecific hybrids. To manipulate parental phenotypic divergence independently of genotype, we also measured the competitive growth of a single interspecific hybrid relative to its parents in 12 different environments. In these assays, we not only identified a strong relationship between parental phenotypic divergence and mid‐parent heterosis as before, but, more tentatively, a weak relationship between phenotypic divergence and best‐parent heterosis, suggesting that complementation of deleterious mutations was not the sole cause of interspecific heterosis. Our results show that mating between different species can be beneficial, and demonstrate that competition assays between parents and offspring are a useful way to study the evolutionary consequences of hybridization.     

Fitness of Crop-Wild Hybrid Sunflower under Competitive Conditions: Implications for Crop-to-Wild Introgression

Understanding the likelihood and extent of introgression of novel alleles in hybrid zones requires comparison of lifetime fitness of parents and hybrid progeny. However, fitness differences among cross types can vary depending on biotic conditions, thereby influencing introgression patterns. Based on past work, we predicted that increased competition would enhance introgression between cultivated and wild sunflower (Helianthus annuus) by reducing fitness advantages of wild plants. To test this prediction, we established a factorial field experiment in Kansas, USA where we monitored the fitness of four cross types (Wild, F₁, F₂, and BC_w hybrids) under different levels of interspecific and intraspecific competition. Intraspecific manipulations consisted both of density of competitors and of frequency of crop-wild hybrids. We recorded emergence of overwintered seeds, survival to reproduction, and numbers of seeds produced per reproductive plant. We also calculated two compound fitness measures: seeds produced per emerged seedling and seeds produced per planted seed. Cross type and intraspecific competition affected emergence and survival to reproduction, respectively. Further, cross type interacted with competitive treatments to influence all other fitness traits. More intense competition treatments, especially related to density of intraspecific competitors, repeatedly reduced the fitness advantage of wild plants when considering seeds produced per reproductive plant and per emerged seedling, and F₂ plants often became indistinguishable from the wilds. Wild fitness remained superior when seedling emergence was also considered as part of fitness, but the fitness of F₂ hybrids relative to wild plants more than quadrupled with the addition of interspecific competitors and high densities of intraspecific competitors. Meanwhile, contrary to prediction, lower hybrid frequency reduced wild fitness advantage. These results emphasize the importance of taking a full life cycle perspective. Additionally, due to effects of exogenous selection, a given hybrid generation may be especially well-suited to hastening introgression under particular environmental conditions.