A tool for rapid, automated characterization of population epigenomics in plants.

Epigenetic variation in plant populations is an important factor in determining phenotype and adaptation to the environment. However, while advances have been made in the molecular and computational methods to analyze the methylation status of a given sample of DNA, tools to profile and compare the methylomes of multiple individual plants or groups of plants at high resolution and low cost are lacking. Here, we describe a computational approach and R package (sounDMR) that leverages the benefits of long read nanopore sequencing to enable robust identification of differential methylation from complex experimental designs, as well as assess the variability within treatment groups and identify individual plants of interest. We demonstrate the utility of this approach by profiling a population of Arabidopsis thaliana exposed to a demethylating agent and identify genomic regions of high epigenetic variability between individuals. Given the low cost of nanopore sequencing devices and the ease of sample preparation, these results show that high resolution epigenetic profiling of plant populations can be made more broadly accessible in plant breeding and biotechnology.

Adaptive and nonadaptive causes of heterogeneity in genetic differentiation across the Mimulus guttatus genome.

Genetic diversity becomes structured among populations over time due to genetic drift and divergent selection. Although population structure is often treated as a uniform underlying factor, recent resequencing studies of wild populations have demonstrated that diversity in many regions of the genome may be structured quite dissimilar to the genome-wide pattern. Here, we explored the adaptive and nonadaptive causes of such genomic heterogeneity using population-level, whole genome resequencing data obtained from annual Mimulus guttatus individuals collected across a rugged environment landscape. We found substantial variation in how genetic differentiation is structured both within and between chromosomes, although, in contrast to other studies, known inversion polymorphisms appear to serve only minor roles in this heterogeneity. In addition, much of the genome can be clustered into eight among-population genetic differentiation patterns, but only two of these clusters are particularly consistent with patterns of isolation by distance. By performing genotype-environment association analysis, we also identified genomic intervals where local adaptation to specific climate factors has accentuated genetic differentiation among populations, and candidate genes in these windows indicate climate adaptation may proceed through changes affecting specialized metabolism, drought resistance, and development. Finally, by integrating our findings with previous studies, we show that multiple aspects of plant reproductive biology may be common targets of balancing selection and that variants historically involved in climate adaptation among populations have probably also fuelled rapid adaptation to microgeographic environmental variation within sites.

Population responses to a historic drought across the range of the common monkeyflower (Mimulus guttatus).

PREMISE: Due to climate change, more frequent and intense periodic droughts are predicted to increasingly pose major challenges to the persistence of plant populations. When a severe drought occurs over a broad geographical region, independent responses by individual populations provide replicated natural experiments for examining the evolution of drought resistance and the potential for evolutionary rescue. METHODS: We used a resurrection approach to examine trait evolution in populations of the common monkeyflower, Mimulus guttatus, exposed to a record drought in California from 2011 to 2017. Specifically, we compared variation in traits related to drought escape and avoidance from seeds collected from 37 populations pre- and post-drought in a common garden. In a parallel experiment, we evaluated fitness in two populations, one which thrived and one which was nearly extirpated during the drought, under well-watered and dry-down conditions. RESULTS: We observed substantial variation among populations in trait evolution. In the subset of populations where phenotypes changed significantly, divergence proceeded along trait correlations with some populations flowering rapidly with less vegetative tissue accumulation and others delaying flowering with greater vegetative tissue accumulation. The degree of trait evolution was only weakly correlated with drought intensity but strongly correlated with initial levels of standing variation. Fitness was higher in the post-drought than pre-drought accessions in both treatments for the thriving population, but lower in both treatments for the nearly extirpated population. CONCLUSIONS: Together, our results indicate that evolutionary responses to drought are context dependent and reflect the standing genetic variation and genetic correlations present within populations.

Empirical patterns of environmental variation favor adaptive transgenerational plasticity.

Effects of parental environment on offspring traits have been well known for decades. Interest in this transgenerational form of phenotypic plasticity has recently surged due to advances in our understanding of its mechanistic basis. Theoretical research has simultaneously advanced by predicting the environmental conditions that should favor the adaptive evolution of transgenerational plasticity. Yet whether such conditions actually exist in nature remains largely unexplored. Here, using long-term climate data, we modeled optimal levels of transgenerational plasticity for an organism with a one-year life cycle at a spatial resolution of 4 km2 across the continental United States. Both annual temperature and precipitation levels were often autocorrelated, but the strength and direction of these autocorrelations varied considerably even among nearby sites. When present, such environmental autocorrelations render offspring environments statistically predictable based on the parental environment, a key condition for the adaptive evolution of transgenerational plasticity. Results of our optimality models were consistent with this prediction: High levels of transgenerational plasticity were favored at sites with strong environmental autocorrelations, and little-to-no transgenerational plasticity was favored at sites with weak or nonexistent autocorrelations. These results are among the first to show that natural patterns of environmental variation favor the evolution of adaptive transgenerational plasticity. Furthermore, these findings suggest that transgenerational plasticity is likely variable in nature, depending on site-specific patterns of environmental variation.

Lagging Adaptation to Climate Supersedes Local Adaptation to Herbivory in an Annual Monkeyflower.

While native populations are often adapted to historical biotic and abiotic conditions at their home site, populations from other locations in the range may be better adapted to current conditions due to changing climates or extreme conditions in a single year. We examine whether local populations of a widespread species maintain a relative advantage over distant populations that have evolved at sites better matching the current climate. Specifically, we grew lines derived from low- and high-elevation annual populations in California and Oregon of the common monkeyflower (Erythranthe guttata) and conducted phenotypic selection analyses in low- and high-elevation common gardens in Oregon to examine relative fitness and the traits mediating relative fitness. Californian low-elevation populations have the highest relative fitness at the low-elevation site, and Californian high-elevation populations have the highest relative fitness at the high-elevation site. Relative fitness differences are mediated by selection for properly timed transitions to flowering, with selection favoring more rapid growth rates at the low-elevation site and greater vegetative biomass prior to flowering at the high-elevation site. Fitness advantages for Californian plants occur despite incurring higher herbivory at both sites than the native Oregonian plants. Our findings suggest that a lag in adaptation causes maladaptation in extreme years that may be more prevalent in future climates, but local populations still have high growth rates and thus are not yet threatened.

Parental experience modifies the Mimulus methylome.

BACKGROUND: Transgenerational plasticity occurs when the environmental experience of an organism modifies the growth and development of its progeny. Leaf damage in Mimulus guttatus exhibits transgenerational plasticity mediated through differential expression of hundreds of genes. The epigenetic mechanisms that facilitate this response have yet to be described. RESULTS: We performed whole genome bisulfite sequencing in the progeny of genetically identical damaged and control plants and developed a pipeline to compare differences in the mean and variance of methylation between treatment groups. We find that parental damage increases the variability of CG and CHG methylation among progeny, but does not alter the overall mean methylation. Instead it has positive effects in some regions and negative in others. We find 3,396 CHH, 203 CG, and 54 CHG Differentially Methylated Regions (DMRs) ranging from tens to thousands of base pairs scattered across the genome. CHG and CHH DMRs tended to overlap with transposable elements. CG DMRs tended to overlap with gene coding regions, many of which were previously found to be differentially expressed. CONCLUSIONS: Genome-wide increases in methylome variation suggest that parental conditions can increase epigenetic diversity in response to stress. Additionally, the potential association between CG DMRs and differentially expressed genes supports the hypothesis that differential methylation is a mechanistic component of transgenerational plasticity in M. guttatus.

piRNAs Are Associated with Diverse Transgenerational Effects on Gene and Transposon Expression in a Hybrid Dysgenic Syndrome of D. virilis.

Sexual reproduction allows transposable elements (TEs) to proliferate, leading to rapid divergence between populations and species. A significant outcome of divergence in the TE landscape is evident in hybrid dysgenic syndromes, a strong form of genomic incompatibility that can arise when (TE) family abundance differs between two parents. When TEs inherited from the father are absent in the mother's genome, TEs can become activated in the progeny, causing germline damage and sterility. Studies in Drosophila indicate that dysgenesis can occur when TEs inherited paternally are not matched with a pool of corresponding TE silencing PIWI-interacting RNAs (piRNAs) provisioned by the female germline. Using the D. virilis syndrome of hybrid dysgenesis as a model, we characterize the effects that divergence in TE profile between parents has on offspring. Overall, we show that divergence in the TE landscape is associated with persisting differences in germline TE expression when comparing genetically identical females of reciprocal crosses and these differences are transmitted to the next generation. Moreover, chronic and persisting TE expression coincides with increased levels of genic piRNAs associated with reduced gene expression. Combined with these effects, we further demonstrate that gene expression is idiosyncratically influenced by differences in the genic piRNA profile of the parents that arise though polymorphic TE insertions. Overall, these results support a model in which early germline events in dysgenesis establish a chronic, stable state of both TE and gene expression in the germline that is maintained through adulthood and transmitted to the next generation. This work demonstrates that divergence in the TE profile is associated with diverse piRNA-mediated transgenerational effects on gene expression within populations.

DNA methylation and gene expression in Mimulus guttatus.

BACKGROUND: The presence of methyl groups on cytosine nucleotides across an organism's genome (methylation) is a major regulator of genome stability, crossing over, and gene regulation. The capacity for DNA methylation to be altered by environmental conditions, and potentially passed between generations, makes it a prime candidate for transgenerational epigenetic inheritance. Here we conduct the first analysis of the Mimulus guttatus methylome, with a focus on the relationship between DNA methylation and gene expression. RESULTS: We present a whole genome methylome for the inbred line Iron Mountain 62 (IM62). DNA methylation varies across chromosomes, genomic regions, and genes. We develop a model that predicts gene expression based on DNA methylation (R(2) = 0.2). Post hoc analysis of this model confirms prior relationships, and identifies novel relationships between methylation and gene expression. Additionally, we find that DNA methylation is significantly depleted near gene transcriptional start sites, which may explain the recently discovered elevated rate of recombination in these same regions. CONCLUSIONS: The establishment here of a reference methylome will be a useful resource for the continued advancement of M. guttatus as a model system. Using a model-based approach, we demonstrate that methylation patterns are an important predictor of variation in gene expression. This model provides a novel approach for differential methylation analysis that generates distinct and testable hypotheses regarding gene expression.

Replicate altitudinal clines reveal that evolutionary flexibility underlies adaptation to drought stress in annual Mimulus guttatus.

Examining how morphology, life history and physiology vary along environmental clines can reveal functional insight into adaptations to climate and thus inform predictions about evolutionary responses to global change. Widespread species occurring over latitudinal and altitudinal gradients in seasonal water availability are excellent systems for investigating multivariate adaptation to drought stress. Under common garden conditions, we characterized variation in 27 traits for 52 annual populations of Mimulus guttatus sampled from 10 altitudinal transects. We also assessed variation in the critical photoperiod for flowering and surveyed neutral genetic markers to control for demography when analyzing clinal patterns. Many drought escape (e.g. flowering time) and drought avoidance (e.g. specific leaf area, succulence) traits exhibited geographic or climatic clines, which often remained significant after accounting for population structure. Critical photoperiod and flowering time in glasshouse conditions followed distinct clinal patterns, indicating different aspects of seasonal phenology confer adaptation to unique agents of selection. Although escape and avoidance traits were negatively correlated range-wide, populations from sites with short growing seasons produced both early flowering and dehydration avoidance phenotypes. Our results highlight how abundant genetic variation in the component traits that build multivariate adaptations to drought stress provides flexibility for intraspecific adaptation to diverse climates.

Gene expression plasticity resulting from parental leaf damage in Mimulus guttatus.

Leaf trichome density in Mimulus guttatus can be altered by the parental environment. In this study, we compared global gene expression patterns in progeny of damaged and control plants. Significant differences in gene expression probably explain the observed trichome response, and identify additional responsive pathways. Using whole transcriptome RNA sequencing, we estimated differential gene expression between isogenic seedlings whose parents had, or had not, been subject to leaf damage. We identified over 900 genes that were differentially expressed in response to parental wounding. These genes clustered into groups involved in cell wall and cell membrane development, stress response pathways, and secondary metabolism. Gene expression is modified as a consequence of the parental environment in a targeted way that probably alters multiple developmental pathways, and may increase progeny fitness if they experience environments similar to that of their parents.