Mutation bias reflects natural selection in Arabidopsis thaliana.

Since the first half of the twentieth century, evolutionary theory has been dominated by the idea that mutations occur randomly with respect to their consequences1. Here we test this assumption with large surveys of de novo mutations in the plant Arabidopsis thaliana. In contrast to expectations, we find that mutations occur less often in functionally constrained regions of the genome-mutation frequency is reduced by half inside gene bodies and by two-thirds in essential genes. With independent genomic mutation datasets, including from the largest Arabidopsis mutation accumulation experiment conducted to date, we demonstrate that epigenomic and physical features explain over 90% of variance in the genome-wide pattern of mutation bias surrounding genes. Observed mutation frequencies around genes in turn accurately predict patterns of genetic polymorphisms in natural Arabidopsis accessions (r = 0.96). That mutation bias is the primary force behind patterns of sequence evolution around genes in natural accessions is supported by analyses of allele frequencies. Finally, we find that genes subject to stronger purifying selection have a lower mutation rate. We conclude that epigenome-associated mutation bias2 reduces the occurrence of deleterious mutations in Arabidopsis, challenging the prevailing paradigm that mutation is a directionless force in evolution.

Fitness effects of mutation in natural populations of Arabidopsis thaliana reveal a complex influence of local adaptation.

Little is empirically known about the contribution of mutations to fitness in natural environments. However, Fisher's Geometric Model (FGM) provides a conceptual foundation to consider the influence of the environment on mutational effects. To quantify mutational properties in the field, we established eight sets of MA lines (7-10 generations) derived from eight founders collected from natural populations of Arabidopsis thaliana from French and Swedish sites, representing the range margins of the species in Europe. We reciprocally planted the MA lines and their founders at French and Swedish sites, allowing us to test predictions of FGM under naturally occurring environmental conditions. The performance of the MA lines relative to each other and to their respective founders confirmed some and contradicted other predictions of the FGM: the contribution of mutation to fitness variance increased when the genotype was in an environment where its fitness was low, that is, in the away environment, but mutations were more likely to be beneficial when the genotype was in its home environment. Consequently, environmental context plays a large role in the contribution of mutations to the evolutionary process and local adaptation does not guarantee that a genotype is at or close to its optimum.

Advancing Science while Training Undergraduates: Recommendations from a Collaborative Biology Research Network.

Biology research is becoming increasingly dependent on large-scale, "big data," networked research initiatives. At the same time, there has been a corresponding effort to expand undergraduate participation in research to benefit student learning and persistence in science. This essay examines the confluence of this trend through eight years of a collaboration within a successful biology research network that explicitly incorporates undergraduates into large-scale scientific research. We draw upon interviews with faculty in this network to consider the interplay of scientific and pedagogical objectives at the heart of this undergraduate-focused network research project. We identify ways that this network has expanded and diversified access to scientific knowledge production for faculty and students and examine a goal conflict that emerged around the dual objectives of mentoring emerging scientists while producing high-quality scientific data for the larger biology community. Based on lessons learned within this network, we provide three recommendations that can support institutions and faculty engaging in networked research projects with undergraduates: (1) establish rigorous protocols to ensure data and database quality, (2) protect personnel time to coordinate network and scientific processes, and (3) select appropriate partners and establish explicit expectations for specific collaborations.

Undergraduates Phenotyping Arabidopsis Knockouts in a Course-Based Undergraduate Research Experience: Exploring Plant Fitness and Vigor Using Quantitative Phenotyping Methods.

We present a curriculum description, an initial student outcome investigation, and sample scientific results for a representative Course-Based Undergraduate Research Experience (CURE) that is part of the "Undergraduates Phenotyping Arabidopsis Knockouts" (unPAK) network. CUREs in the unPAK network characterize quantitative phenotypes of the model plant Arabidopsis from across environments to uncover connections between genotype and phenotype. Students in unPAK CUREs grow plants in a replicated block design and make quantitative measurements throughout the semester. This CURE enables students to answer plant science questions that draw from fields such as environmental science, genetics, ecology, and evolution. Findings indicate that this experience provides students with opportunities to make relevant scientific discoveries. Eighty percent of student datasets produced from the CURE met criteria for inclusion in the project database, indicative of student learning in data collection and analysis of quantitative plant traits. Student datasets uncovered novel effects of mutation on plant form. In addition, students' science self-efficacy increased as a result of course participation, and faculty feedback on course implementation was positive. We present unPAK as a new network that supports CUREs and research experiences focused on collecting biological data made publicly available to the scientific community. The unPAK CUREs can be tailored to address instructor interests or pedagogical needs while involving students in research investigating quantitative plant phenotypes.

Distributed phenomics with the unPAK project reveals the effects of mutations.

Determining how genes are associated with traits in plants and other organisms is a major challenge in modern biology. The unPAK project - undergraduates phenotyping Arabidopsis knockouts - has generated phenotype data for thousands of non-lethal insertion mutation lines within a single Arabidopsis thaliana genomic background. The focal phenotypes examined by unPAK are complex macroscopic fitness-related traits, which have ecological, evolutionary and agricultural importance. These phenotypes are placed in the context of the wild-type and also natural accessions (phytometers), and standardized for environmental differences between assays. Data from the unPAK project are used to describe broad patterns in the phenotypic consequences of insertion mutation, and to identify individual mutant lines with distinct phenotypes as candidates for further study. Inclusion of undergraduate researchers is at the core of unPAK activities, and an important broader impact of the project is providing students an opportunity to obtain research experience.

Fine-Grained Analysis of Spontaneous Mutation Spectrum and Frequency in Arabidopsis thaliana.

Mutations are the ultimate source of all genetic variation. However, few direct estimates of the contribution of mutation to molecular genetic variation are available. To address this issue, we first analyzed the rate and spectrum of mutations in the Arabidopsis thaliana reference accession after 25 generations of single-seed descent. We then compared the mutation profile in these mutation accumulation (MA) lines against genetic variation observed in the 1001 Genomes Project. The estimated haploid single nucleotide mutation (SNM) rate for A. thaliana is 6.95 × 10-9 (SE ± 2.68 × 10-10) per site per generation, with SNMs having higher frequency in transposable elements (TEs) and centromeric regions. The estimated indel mutation rate is 1.30 × 10-9 (±1.07 × 10-10) per site per generation, with deletions being more frequent and larger than insertions. Among the 1694 unique SNMs identified in the MA lines, the positions of 389 SNMs (23%) coincide with biallelic SNPs from the 1001 Genomes population, and in 289 (17%) cases the changes are identical. Of the 329 unique indels identified in the MA lines, 96 (29%) overlap with indels from the 1001 Genomes dataset, and 16 indels (5% of the total) are identical. These overlap frequencies are significantly higher than expected, suggesting that de novo mutations are not uniformly distributed and arise at polymorphic sites more frequently than assumed. These results suggest that high mutation rate potentially contributes to high polymorphism and low mutation rate to reduced polymorphism in natural populations providing insights of mutational inputs in generating natural genetic diversity.

Quantifying natural seasonal variation in mutation parameters with mutation accumulation lines.

Mutations create novel genetic variants, but their contribution to variation in fitness and other phenotypes may depend on environmental conditions. Furthermore, natural environments may be highly heterogeneous. We assessed phenotypes associated with survival and reproductive success in over 30,000 plants representing 100 mutation accumulation lines of Arabidopsis thaliana across four temporal environments at a single field site. In each of the four assays, environmental variance was substantially larger than mutational variance. For some traits, whether mutational variance was significantly varied between seasons. The founder genotype had mean trait values near the mean of the distribution of the mutation accumulation lines in all field experiments. New mutations also contributed more phenotypic variation than would be predicted, given phenotypic and sequence-level divergence among natural populations of A. thaliana. The combination of large environmental variance with a mean effect of mutation near zero suggests that mutations could contribute substantially to standing genetic variation.

Distributions of Mutational Effects and the Estimation of Directional Selection in Divergent Lineages of Arabidopsis thaliana.

Mutations are crucial to evolution, providing the ultimate source of variation on which natural selection acts. Due to their key role, the distribution of mutational effects on quantitative traits is a key component to any inference regarding historical selection on phenotypic traits. In this paper, we expand on a previously developed test for selection that could be conducted assuming a Gaussian mutation effect distribution by developing approaches to also incorporate any of a family of heavy-tailed Laplace distributions of mutational effects. We apply the test to detect directional natural selection on five traits along the divergence of Columbia and Landsberg lineages of Arabidopsis thaliana, constituting the first test for natural selection in any organism using quantitative trait locus and mutation accumulation data to quantify the intensity of directional selection on a phenotypic trait. We demonstrate that the results of the test for selection can depend on the mutation effect distribution specified. Using the distributions exhibiting the best fit to mutation accumulation data, we infer that natural directional selection caused divergence in the rosette diameter and trichome density traits of the Columbia and Landsberg lineages.

Field measurements of genotype by environment interaction for fitness caused by spontaneous mutations in Arabidopsis thaliana.

As the ultimate source of genetic diversity, spontaneous mutation is critical to the evolutionary process. The fitness effects of spontaneous mutations are almost always studied under controlled laboratory conditions rather than under the evolutionarily relevant conditions of the field. Of particular interest is the conditionality of new mutations-that is, is a new mutation harmful regardless of the environment in which it is found? In other words, what is the extent of genotype-environment interaction for spontaneous mutations? We studied the fitness effects of 25 generations of accumulated spontaneous mutations in Arabidopsis thaliana in two geographically widely separated field environments, in Michigan and Virginia. At both sites, mean total fitness of mutation accumulation lines exceeded that of the ancestors, contrary to the expected decrease in the mean due to new mutations but in accord with prior work on these MA lines. We observed genotype-environment interactions in the fitness effects of new mutations, such that the effects of mutations in Michigan were a poor predictor of their effects in Virginia and vice versa. In particular, mutational variance for fitness was much larger in Virginia compared to Michigan. This strong genotype-environment interaction would increase the amount of genetic variation maintained by mutation-selection balance.

Extensive variation in cadmium tolerance and accumulation among populations of Chamaecrista fasciculata.

Plant populations may vary substantially in their tolerance for and accumulation of heavy metals, and assessment of this variability is important when selecting species to use in restoration or phytoremediation projects. We examined the population variation in cadmium tolerance and accumulation in a leguminous pioneer species native to the eastern United States, the partridge pea (Chamaecrista fasciculata). We assayed growth, reproduction and patterns of cadmium accumulation in six populations of C. fasciculata grown on a range of cadmium-contaminated soils. In general, C. fasciculata exhibited tolerance in low to moderate soil cadmium concentrations. Both tolerance and accumulation patterns varied across populations. C. fasciculata exhibited many characteristics of a hyperaccumulator species, with high cadmium uptake in shoots and roots. However, cadmium was excluded from extrafloral nectar. As a legume with tolerance for moderate cadmium contamination, C. fasciculata has potential for phytoremediation. However, our findings also indicate the importance of considering the effects of genetic variation on plant performance when screening plant populations for utilization in remediation and restoration activities. Also, there is potential for cadmium contamination to affect other species through contamination of leaves, fruits, flowers, pollen and root nodules.

Extensive phenotypic variation among allelic T-DNA inserts in Arabidopsis thaliana.

T-DNA insertion mutants are a tool used widely in Arabidopsis thaliana to disrupt gene function. We phenotyped multiple homozygous T-DNA A. thaliana mutants at each of two loci (AT1G11060 and AT4G00210). We measured life history traits, including germination, size at reproduction and fruit production. Allelic T-DNA lines differed for most traits at AT1G11060 but not at AT4G00210. However, insertions in exons differed from other insertion positions in AT4G00210 but not in AT1G11060. We found evidence for additional insertions in approximately half of the lines, but found few phenotypic consequences. In general, our results suggest that a cautious interpretation of T-DNA phenotypes is warranted.

Fitness of Arabidopsis thaliana mutation accumulation lines whose spontaneous mutations are known.

Despite the fundamental importance of mutation to the evolutionary process, we have little knowledge of the direct consequences of specific spontaneous mutations to the fitness of the organism. Combining results of whole-genome sequencing with repeated field assays of survival and reproduction, we quantify the combined effects on fitness of spontaneous mutations identified in Arabidopsis thaliana. We demonstrate that the effects are beneficial, deleterious, or neutral depending on the environmental context. Some lines, bearing mutations disrupting known loci, differ strongly in fitness from the founder or premutation genotype. Those effects vary across environments, for example, a line with a major deletion spanning a transcription factor gene expressed lower fitness than the founder under most conditions but exceeded the founder's fitness in one environment. The large contribution of genotype by environment interaction (G × E) to mutation effects on fitness implies spatial and/or temporal variation in selection on new mutations and could contribute to the maintenance of standing genetic variation.

Birth, death and subfunctionalization in the Arabidopsis genome.

Arabidopsis thaliana is now a model system, not just for plant biology but also for comparative genomics. The completion of the sequences of two closely related species, Arabidopsis lyrata and Brassica rapa, is complemented by genomic comparisons among A. thaliana accessions and mutation accumulation lines. Together these genomic data document the birth of new genes via gene duplication, transposon exaptation and de novo formation of new genes from noncoding sequence. Most novel loci exhibit low expression, and are undergoing pseudogenization or subfunctionalization. Comparatively, A. thaliana has lost large amounts of sequence through deletion, particularly of transposable elements. Intraspecific genomic variation indicates high rates of deletion mutations and deletion polymorphisms across accessions, shedding light on the history of Arabidopsis genome architecture.

Spontaneous mutation parameters for Arabidopsis thaliana measured in the wild.

Mutations are the ultimate source of genetic diversity and their contributions to evolutionary process depend critically on their rate and their effects on traits, notably fitness. Mutation rate and mutation effect can be measured simultaneously through the use of mutation accumulation lines, and previous mutation accumulation studies measuring these parameters have been performed in laboratory conditions. However, estimation of mutation parameters for fitness in wild populations requires assays in environments where mutations are exposed to natural selection and natural environmental variation. Here we quantify mutation parameters in both the wild and greenhouse environments using 100 25th generation Arabidopsis thaliana mutation accumulation lines. We found significantly greater mutational variance and a higher mutation rate for fitness under field conditions relative to greenhouse conditions. However, our field estimates were low when scaled to natural environmental variation. Many of the mutation accumulation lines have increased fitness, counter to the expectation that nearly all mutations decrease fitness. A high mutation rate and a low mutational contribution to phenotypic variation may explain observed levels of natural genetic variation. Our findings indicate that mutation parameters are not fixed, but are variables whose values may reflect the specific environment in which mutations are tested.

Testing for adaptation to climate in Arabidopsis thaliana: a calibrated common garden approach.

BACKGROUND AND AIMS: A recent method used to test for local adaptation is a common garden experiment where analyses are calibrated to the environmental conditions of the garden. In this study the calibrated common garden approach is used to test for patterns of adaptation to climate in accessions of Arabidopsis thaliana. METHODS: Seedlings from 21 accessions of A. thaliana were planted outdoors in College Park, MD, USA, and development was monitored during the course of a growing season. ANOVA and multiple regression analysis were used to determine if development traits were significant predictors of plant success. Previously published data relating to accessional differences in genetic and physiological characters were also examined. Historical records of climate were used to evaluate whether properties of the site of origin of an accession affected the fitness of plants in a novel environment. KEY RESULTS: By calibrating the analysis to the climatic conditions of the common garden site, performance differences were detected among the accessions consistent with a pattern of adaptation to latitude and climatic conditions. Relatively higher accession fitness was predicted by a latitude and climatic history similar to that of College Park in April and May during the main growth period of this experiment. The climatic histories of the accessions were better predictors of performance than many of the life-history and growth measures taken during the experiment. CONCLUSIONS: It is concluded that the calibrated common garden experiment can detect local adaptation and guide subsequent reciprocal transplant experiments.

Pollination and seed predation by moths on Silene and allied Caryophyllaceae: evaluating a model system to study the evolution of mutualisms.

Nursery pollinators, and the plants they use as hosts for offspring development, function as exemplary models of coevolutionary mutualism. The two pre-eminent examples--fig wasps and yucca moths--show little variation in the interaction: the primary pollinator is an obligate mutualist. By contrast, nursery pollination of certain Caryophyllaceae, including Silene spp., by two nocturnal moth genera, Hadena and Perizoma, ranges from antagonistic to potentially mutualistic, offering an opportunity to test hypotheses about the factors that promote or discourage the evolution of mutualism. Here, we review nursery pollination and host-plant interactions in over 30 caryophyllaceous plants, based on published studies and a survey of researchers investigating pollination, seed predation, and moth morphology and behavior. We detected little direct evidence of mutualism in these moth-plant interactions, but found traits and patterns in both that are nonetheless consistent with the evolution of mutualism and merit further attention.

Pathway length and evolutionary constraint in amino acid biosynthesis.

The evolutionary properties of a metabolic network may be determined by the topology of the network. One attribute of pathways that make up the network is the number of enzymatic steps between initial substrates and final products. To determine the effect of pathway length on evolutionary lability of pathway structure, we examined amino acid biosynthetic pathways across 48 sequenced organisms. We demonstrate that longer pathways exhibit lower rates of change in pathway structure than shorter pathways. This finding suggests that increasing complexity may increase constraint on evolutionary change.

Natural selection on extrafloral nectar production in Chamaecrista fasciculata: the costs and benefits of a mutualism trait.

Cost-benefit models of the evolution of mutualism predict that the current state of mutualism results from trade-offs between fitness costs of mutualist traits and the fitness benefits of association. We test the assumptions of such models by measuring patterns of natural selection on a mutualist trait, extrafloral nectar production in Chamaecrista fasciculata. Selection was measured on plants from which ants had been excluded (removing the mutualist benefit of the trait), from which all insects had been excluded (removing costs of herbivory in addition to mutualist benefits), and unmanipulated plants (where both costs and benefits were present). Selection analysis based on half-sibling-mean regressions of fitness on the trait revealed no evidence of costs of extrafloral nectar production in the absence of all insects or in the absence of ants. However, examination of the selective surfaces for these treatments suggest that costs of nectar production may exist and are exacerbated by the presence of herbivory. In the presence of ants, natural selection favors high extrafloral nectar production, consistent with a fitness benefit to this mutualist trait in the presence of the mutualist partner. In this study, the interaction of costs and benefits did not produce an evolutionary optimum for the trait within the range of variation observed, suggesting that application of a cost-benefit framework to this trait will benefit from considering the influence of temporal and spatial variation on the quality of costs and benefits.