The quantitative genetics of gene expression in Mimulus guttatus.

Gene expression can be influenced by genetic variants that are closely linked to the expressed gene (cis eQTLs) and variants in other parts of the genome (trans eQTLs). We created a multiparental mapping population by sampling genotypes from a single natural population of Mimulus guttatus and scored gene expression in the leaves of 1,588 plants. We find that nearly every measured gene exhibits cis regulatory variation (91% have FDR < 0.05). cis eQTLs are usually allelic series with three or more functionally distinct alleles. The cis locus explains about two thirds of the standing genetic variance (on average) but varies among genes and tends to be greatest when there is high indel variation in the upstream regulatory region and high nucleotide diversity in the coding sequence. Despite mapping over 10,000 trans eQTL / affected gene pairs, most of the genetic variance generated by trans acting loci remains unexplained. This implies a large reservoir of trans acting genes with subtle or diffuse effects. Mapped trans eQTLs show lower allelic diversity but much higher genetic dominance than cis eQTLs. Several analyses also indicate that trans eQTLs make a substantial contribution to the genetic correlations in expression among different genes. They may thus be essential determinants of "gene expression modules," which has important implications for the evolution of gene expression and how it is studied by geneticists.

A few essential genetic loci distinguish Penstemon species with flowers adapted to pollination by bees or hummingbirds.

In the formation of species, adaptation by natural selection generates distinct combinations of traits that function well together. The maintenance of adaptive trait combinations in the face of gene flow depends on the strength and nature of selection acting on the underlying genetic loci. Floral pollination syndromes exemplify the evolution of trait combinations adaptive for particular pollinators. The North American wildflower genus Penstemon displays remarkable floral syndrome convergence, with at least 20 separate lineages that have evolved from ancestral bee pollination syndrome (wide blue-purple flowers that present a landing platform for bees and small amounts of nectar) to hummingbird pollination syndrome (bright red narrowly tubular flowers offering copious nectar). Related taxa that differ in floral syndrome offer an attractive opportunity to examine the genomic basis of complex trait divergence. In this study, we characterized genomic divergence among 229 individuals from a Penstemon species complex that includes both bee and hummingbird floral syndromes. Field plants are easily classified into species based on phenotypic differences and hybrids displaying intermediate floral syndromes are rare. Despite unambiguous phenotypic differences, genome-wide differentiation between species is minimal. Hummingbird-adapted populations are more genetically similar to nearby bee-adapted populations than to geographically distant hummingbird-adapted populations, in terms of genome-wide dXY. However, a small number of genetic loci are strongly differentiated between species. These approximately 20 "species-diagnostic loci," which appear to have nearly fixed differences between pollination syndromes, are sprinkled throughout the genome in high recombination regions. Several map closely to previously established floral trait quantitative trait loci (QTLs). The striking difference between the diagnostic loci and the genome as whole suggests strong selection to maintain distinct combinations of traits, but with sufficient gene flow to homogenize the genomic background. A surprisingly small number of alleles confer phenotypic differences that form the basis of species identity in this species complex.

Predicting evolutionary change at the DNA level in a natural Mimulus population.

Evolution by natural selection occurs when the frequencies of genetic variants change because individuals differ in Darwinian fitness components such as survival or reproductive success. Differential fitness has been demonstrated in field studies of many organisms, but it remains unclear how well we can quantitatively predict allele frequency changes from fitness measurements. Here, we characterize natural selection on millions of Single Nucleotide Polymorphisms (SNPs) across the genome of the annual plant Mimulus guttatus. We use fitness estimates to calibrate population genetic models that effectively predict allele frequency changes into the next generation. Hundreds of SNPs experienced "male selection" in 2013 with one allele at each SNP elevated in frequency among successful male gametes relative to the entire population of adults. In the following generation, allele frequencies at these SNPs consistently shifted in the predicted direction. A second year of study revealed that SNPs had effects on both viability and reproductive success with pervasive trade-offs between fitness components. SNPs favored by male selection were, on average, detrimental to survival. These trade-offs (antagonistic pleiotropy and temporal fluctuations in fitness) may be essential to the long-term maintenance of alleles. Despite the challenges of measuring selection in the wild, the strong correlation between predicted and observed allele frequency changes suggests that population genetic models have a much greater role to play in forward-time prediction of evolutionary change.

Multiplexed shotgun genotyping (MSG) data resolve phylogenetic relationships within and among archipelagos in Macaronesian Tolpis.

PREMISE: Plants endemic to oceanic archipelagos are suitable for studying evolution, being isolated on substrates of different ages. Evolution has been recent, rendering traditionally employed sequences insufficiently variable for resolving relationships. This study includes sampling in the genus Tolpis (Asteraceae) from the Azores, Madeira, and Cape Verde, and expands upon an earlier study demonstrating the efficacy of multiplexed shotgun genotyping (MSG) for resolving relationships in Canarian Tolpis. METHODS: Genomic libraries for 90 accessions of Tolpis and two from the outgroup were generated for genotyping individuals using MSG. Loci were de novo assembled with iPyrad, which clusters MSG loci within and between samples. A maximum likelihood phylogeny was generated with RAxML. Ancestral area reconstruction was inferred using R package BioGeoBEARS. RESULTS: MSG data recovered a highly resolved phylogeny from population to inter-archipelago levels. Ancestral area reconstruction provided biogeographic hypotheses for the radiation of Macaronesian Tolpis. CONCLUSIONS: Four major clades were resolved. The Madeiran endemic T. macrorhiza is sister to other Tolpis. Species from the Canaries, Cape Verdes, and the continent are sister to T. succulenta from Madeira, which has a sister subclade of Azorean populations composed of T. succulenta and T. azorica. Population-level resolution suggests unrecognized taxa on several archipelagos. Ancestral reconstruction suggests initial dispersal from the continent to Madeira, with dispersal to the Azores, then dispersal from Madeira to the Canary Islands, with both subsequent dispersal to the Cape Verdes and back-dispersal to the continent. Single-island radiations and inter-island dispersal are implicated in divergence in Macaronesian Tolpis.

The promise and deceit of genomic selection component analyses.

Selection component analyses (SCA) relate individual genotype to fitness components such as viability, fecundity and mating success. SCA are based on population genetic models and yield selection estimates directly in terms of predicted allele frequency change. This paper explores the statistical properties of gSCA: experiments that apply SCA to genome-wide scoring of SNPs in field sampled individuals. Computer simulations indicate that gSCA involving a few thousand genotyped samples can detect allele frequency changes of the magnitude that has been documented in field experiments on diverse taxa. To detect selection, imprecise genotyping from low-level sequencing of large samples of individuals provides much greater power than precise genotyping of smaller samples. The simulations also demonstrate the efficacy of 'haplotype matching', a method to combine information from a limited collection of whole genome sequence (the reference panel) with the much larger sample of field individuals that are measured for fitness. Pooled sequencing is demonstrated as another way to increase statistical power. Finally, I discuss the interpretation of selection estimates in relation to the Beavis effect, the overestimation of selection intensities at significant loci.

GOOGA: A platform to synthesize mapping experiments and identify genomic structural diversity.

Understanding genomic structural variation such as inversions and translocations is a key challenge in evolutionary genetics. We develop a novel statistical approach to comparative genetic mapping to detect large-scale structural mutations from low-level sequencing data. The procedure, called Genome Order Optimization by Genetic Algorithm (GOOGA), couples a Hidden Markov Model with a Genetic Algorithm to analyze data from genetic mapping populations. We demonstrate the method using both simulated data (calibrated from experiments on Drosophila melanogaster) and real data from five distinct crosses within the flowering plant genus Mimulus. Application of GOOGA to the Mimulus data corrects numerous errors (misplaced sequences) in the M. guttatus reference genome and confirms or detects eight large inversions polymorphic within the species complex. Finally, we show how this method can be applied in genomic scans to improve the accuracy and resolution of Quantitative Trait Locus (QTL) mapping.

Genome-wide genotyping estimates mating system parameters and paternity in the island species Tolpis succulenta.

PREMISE: The mating system has profound consequences, not only for ecology and evolution, but also for the conservation of threatened or endangered species. Unfortunately, small populations are difficult to study owing to limits on sample size and genetic marker diversity. Here, we estimated mating system parameters in three small populations of an island plant using genomic genotyping. Although self-incompatible (SI) species are known to often set some self-seed, little is known about how "leaky SI" affects selfing rates in nature or the role that multiple paternity plays in small populations. METHODS: We generalized the BORICE mating system program to determine the siring pattern within maternal families. We applied this algorithm to maternal families from three populations of Tolpis succulenta from Madeira Island and genotyped the progeny using RADseq. We applied BORICE to estimate each individual offspring as outcrossed or selfed, the paternity of each outcrossed offspring, and the level of inbreeding of each maternal plant. RESULTS: Despite a functional self-incompatibility system, these data establish T. succulenta as a pseudo-self-compatible (PSC) species. Two of 75 offspring were strongly indicated as products of self-fertilization. Despite selfing, all adult maternal plants were fully outbred. There was high differentiation among and low variation within populations, consistent with a history of genetic isolation of these small populations. There were generally multiple sires per maternal family. Twenty-two percent of sib contrasts (between outcrossed offspring within maternal families) shared the same sire. CONCLUSIONS: Genome-wide genotyping, combined with appropriate analytical methods, enables estimation of mating system and multiple paternity in small populations. These data address questions about the evolution of reproductive traits and the conservation of threatened populations.

Extreme copy number variation at a tRNA ligase gene affecting phenology and fitness in yellow monkeyflowers.

Copy number variation (CNV) is a major part of the genetic diversity segregating within populations, but remains poorly understood relative to single nucleotide variation. Here, we report on a tRNA ligase gene (Migut.N02091; RLG1a) exhibiting unprecedented, and fitness-relevant, CNV within an annual population of the yellow monkeyflower Mimulus guttatus. RLG1a variation was associated with multiple traits in pooled population sequencing (PoolSeq) scans of phenotypic and phenological cohorts. Resequencing of inbred lines revealed intermediate-frequency three-copy variants of RLG1a (trip+; 5/35 = 14%), and trip+ lines exhibited elevated RLG1a expression under multiple conditions. trip+ carriers, in addition to being over-represented in late-flowering and large-flowered PoolSeq populations, flowered later under stressful conditions in a greenhouse experiment (p < 0.05). In wild population samples, we discovered an additional rare RLG1a variant (high+) that carries 250-300 copies of RLG1a totalling ~5.7 Mb (20-40% of a chromosome). In the progeny of a high+ carrier, Mendelian segregation of diagnostic alleles and qPCR-based copy counts indicate that high+ is a single tandem array unlinked to the single-copy RLG1a locus. In the wild, high+ carriers had highest fitness in two particularly dry and/or hot years (2015 and 2017; both p < 0.01), while single-copy individuals were twice as fecund as either CNV type in a lush year (2016: p < 0.005). Our results demonstrate fluctuating selection on CNVs affecting phenological traits in a wild population, suggest that plant tRNA ligases mediate stress-responsive life-history traits, and introduce a novel system for investigating the molecular mechanisms of gene amplification.

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.

Selfing Can Facilitate Transitions between Pollination Syndromes.

Pollinator-mediated selection on plants can favor transitions to a new pollinator depending on the relative abundances and efficiencies of pollinators present in the community. A frequently observed example is the transition from bee pollination to hummingbird pollination. We present a population genetic model that examines whether the ability to inbreed can influence evolutionary change in traits that underlie pollinator attraction. We find that a transition to a more efficient but less abundant pollinator is favored under a broadened set of ecological conditions if plants are capable of delayed selfing rather than obligately outcrossing. Delayed selfing allows plants carrying an allele that attracts the novel pollinator to reproduce even when this pollinator is rare, providing reproductive assurance. In addition, delayed selfing weakens the effects of Haldane's sieve by increasing the fixation probability for recessive alleles that confer adaptation to the new pollinator. Our model provides novel insight into the paradoxical abundance of recessive mutations in adaptation to hummingbird attraction. It further predicts that transitions to efficient but less abundant pollinators (such as hummingbirds in certain communities) should disproportionately occur in self-compatible lineages. Currently available mating system data sets are consistent with this prediction, and we suggest future areas of research that will enable a rigorous test of this theory.

Epistasis Is a Major Determinant of the Additive Genetic Variance in Mimulus guttatus.

The influence of genetic interactions (epistasis) on the genetic variance of quantitative traits is a major unresolved problem relevant to medical, agricultural, and evolutionary genetics. The additive genetic component is typically a high proportion of the total genetic variance in quantitative traits, despite that underlying genes must interact to determine phenotype. This study estimates direct and interaction effects for 11 pairs of Quantitative Trait Loci (QTLs) affecting floral traits within a single population of Mimulus guttatus. With estimates of all 9 genotypes for each QTL pair, we are able to map from QTL effects to variance components as a function of population allele frequencies, and thus predict changes in variance components as allele frequencies change. This mapping requires an analytical framework that properly accounts for bias introduced by estimation errors. We find that even with abundant interactions between QTLs, most of the genetic variance is likely to be additive. However, the strong dependency of allelic average effects on genetic background implies that epistasis is a major determinant of the additive genetic variance, and thus, the population's ability to respond to selection.

The genetic breakdown of sporophytic self-incompatibility in Tolpis coronopifolia (Asteraceae).

Angiosperm diversity has been shaped by mating system evolution, with the most common transition from outcrossing to self-fertilizing. To investigate the genetic basis of this transition, we performed crosses between two species endemic to the Canary Islands, the self-compatible (SC) species Tolpis coronopifolia and its self-incompatible (SI) relative Tolpis santosii. We scored self-compatibility as self-seed set of recombinant plants within two F2 populations. To map and genetically characterize the breakdown of SI, we built a draft genome sequence of T. coronopifolia, genotyped F2 plants using multiplexed shotgun genotyping (MSG), and located MSG markers to the genome sequence. We identified a single quantitative trait locus (QTL) that explains nearly all variation in self-seed set in both F2 populations. To identify putative causal genetic variants within the QTL, we performed transcriptome sequencing on mature floral tissue from both SI and SC species, constructed a transcriptome for each species, and then located each predicted transcript to the T. coronopifolia genome sequence. We annotated each predicted gene within the QTL and found two strong candidates for SI breakdown. Each gene has a coding sequence insertion/deletion mutation within the SC species that produces a truncated protein. Homologs of each gene have been implicated in pollen development, pollen germination, and pollen tube growth in other species.

Population structure and local selection yield high genomic variation in Mimulus guttatus.

Across western North America, Mimulus guttatus exists as many local populations adapted to site-specific environmental challenges. Gene flow between locally adapted populations will affect genetic diversity both within demes and across the larger metapopulation. Here, we analyse 34 whole-genome sequences from the intensively studied Iron Mountain population (IM) in conjunction with sequences from 22 Mimulus individuals sampled from across western North America. Three striking features of these data address hypotheses about migration and selection in a locally adapted population. First, we find very high levels of intrapopulation polymorphism (synonymous π = 0.033). Variation outside of genes is likely even higher but difficult to estimate because excessive divergence reduces the efficiency of read mapping. Second, IM exhibits a significantly positive genomewide average for Tajima's D. This indicates allele frequencies are typically more intermediate than expected from neutrality, opposite the pattern observed in many other species. Third, IM exhibits a distinctive haplotype structure with a genomewide excess of positive associations between rarer alleles at linked loci. This suggests an important effect of gene flow from other Mimulus populations, although a residual effect of population founding might also contribute. The combination of multiple analyses, including a novel tree-based analytic method, illustrates how the balance of local selection, limited dispersal and metapopulation dynamics manifests across the genome. The overall genomic pattern of sequence diversity suggests successful gene flow of divergent immigrant genotypes into IM. However, many loci show patterns indicative of local adaptation, particularly at SNPs associated with chromosomal inversions.

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.

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.

Multiplexed-shotgun-genotyping data resolve phylogeny within a very recently derived insular lineage.

PREMISE OF THE STUDY: Endemic plants on oceanic islands have long served as model systems for studying patterns and processes of evolution. However, phylogenetic studies of island plants frequently illustrate a decoupling of molecular divergence and ecological/morphological diversity, resulting in phylogenies lacking the resolution required to interpret patterns of evolution in a phylogenetic context. The current study uses the primarily Macaronesian flowering plant genus Tolpis to illustrate the utility of multiplexed shotgun genotyping (MSG) for resolving relationships at relatively deep (among archipelagos) and very shallow (within archipelagos) nodes in this small, yet diverse insular plant lineage that had not been resolved with other molecular markers. METHODS: Genomic libraries for 27 accessions of Macaronesian Tolpis were generated for genotyping individuals using MSG, a form of reduced-representation sequencing, similar to restriction-site-associated DNA markers (RADseq). The resulting data files were processed using the program pyRAD, which clusters MSG loci within and between samples. Phylogenetic analyses of the aligned data matrix were conducted using RAxML. KEY RESULTS: Analysis of MSG data recovered a highly resolved phylogeny with generally strong support, including the first robust inference of relationships within the highly diverse Canary Island clade of Tolpis. CONCLUSIONS: The current study illustrates the utility of MSG data for resolving relationships in lineages that have undergone recent, rapid diversification resulting in extensive ecological and morphological diversity. We suggest that a similar approach may prove generally useful for other rapid plant radiations where resolving phylogeny has been difficult.

Naturally segregating loci exhibit epistasis for fitness.

The extent to which gene interaction or epistasis contributes to fitness variation within populations remains poorly understood, despite its importance to a myriad of evolutionary questions. Here, we report a multi-year field study estimating fitness of Mimulus guttatus genetic lines in which pairs of naturally segregating loci exist in an otherwise uniform background. An allele at QTL x5b-a locus originally mapped for its effect on flower size-positively affects survival if combined with one genotype at quantitative trait locus x10a (aa) but has negative effects when combined with the other genotypes (Aa and AA). The viability differences between genotypes parallel phenotypic differences for the time and node at which a plant flowers. Viability is negatively correlated with fecundity across genotypes, indicating antagonistic pleiotropy for fitness components. This trade-off reduces the genetic variance for total fitness relative to the individual fitness components and thus may serve to maintain variation. Additionally, we find that the effects of each locus and their interaction often vary with the environment.

On the importance of balancing selection in plants.

Balancing selection refers to a variety of selective regimes that maintain advantageous genetic diversity within populations. We review the history of the ideas regarding the types of selection that maintain such polymorphism in flowering plants, notably heterozygote advantage, negative frequency-dependent selection, and spatial heterogeneity. One shared feature of these mechanisms is that whether an allele is beneficial or detrimental is conditional on its frequency in the population. We highlight examples of balancing selection on a variety of discrete traits. These include the well-referenced case of self-incompatibility and recent evidence from species with nuclear-cytoplasmic gynodioecy, both of which exhibit trans-specific polymorphism, a hallmark of balancing selection. We also discuss and give examples of how spatial heterogeneity in particular, which is often thought unlikely to allow protected polymorphism, can maintain genetic variation in plants (which are rooted in place) as a result of microhabitat selection. Lastly, we discuss limitations of the protected polymorphism concept for quantitative traits, where selection can inflate the genetic variance without maintaining specific alleles indefinitely. We conclude that while discrete-morph variation provides the most unambiguous cases of protected polymorphism, they represent only a fraction of the balancing selection at work in plants.