Matthew A. Streisfeld

POPULATION GENETICS, PLEIOTROPY, AND THE PREFERENTIAL FIXATION OF MUTATIONS DURING ADAPTIVE EVOLUTION

Ongoing debate centers on whether certain types of mutations are fixed preferentially during adaptive evolution. Although there has been much discussion, no quantitative framework currently exists to test for these biases. Here, we describe a method for distinguishing between the two processes that likely account for biased rates of substitution: variation in mutation rates and variation in the probability that a mutation becomes fixed once it arises. We then use this approach to examine the type and magnitude of these biases during evolutionary transitions across multiple scales: those involving repeated origins of individual traits (flower color change), and transitions involving broad suites of traits (morphological and physiological trait evolution in plants and animals). We show that fixation biases can be strong at both levels of comparison, likely due to differences in the magnitude of deleterious pleiotropy associated with alternative mutation categories. However, we also show that the scale at which these comparisons are made greatly influences the results, as broad comparisons that simultaneously analyze mutliple traits obscure heterogeneity in the direction and magnitude of these biases. We conclude that preferential fixation of mutations likely is common in nature, but should be studied on a trait‐by‐trait basis.

Altered trans-Regulatory Control of Gene Expression in Multiple Anthocyanin Genes Contributes to Adaptive Flower Color Evolution in Mimulus aurantiacus

A fundamental goal in evolutionary biology is to identify the molecular changes responsible for adaptive evolution. In this study, we describe a genetic analysis to determine whether the molecular changes contributing to adaptive flower color divergence in Mimulus aurantiacus affect gene expression or enzymatic activity. High performance liquid chromatography analysis confirms that flower color differences are caused by the presence versus absence of anthocyanin pigments. Cosegregation analysis and in vitro enzymatic assays rule out mutations that affect enzymatic function in the anthocyanin pathway genes. By contrast, cosegregation of gene expression with flower color suggests that tissue-specific differences in pigment production are caused by the coordinated regulatory control of three anthocyanin pathway genes. We provide evidence indicating that these expression differences are caused by a locus that acts in trans- and explains 45% of the phenotypic variance in flower color. A second locus with sequence similarity to the R2R3 MYB family of transcription factors explains 9% of the variation but does so in a complex fashion. These results demonstrate one of only two examples where we have clear evidence of both the adaptive nature of a flower color transition and evidence for its genetic basis. In both cases, mutations appear to affect expression of the anthocyanin structural genes. Future studies will allow us to determine whether these differences represent a real bias in favor of mutations that affect gene expression.

Genetic changes contributing to the parallel evolution of red floral pigmentation among Ipomoea species

The repeated, independent evolution of phenotypic traits reflects adaptation to similar selective pressures. In some circumstances, parallel phenotypic evolution has a common genetic basis. Here, we investigate the types of genetic change responsible for the repeated evolution of red flowers among Ipomoea species. We identified three independent transitions from cyanidin- (blue/purple) to pelargonidin-type (red) anthocyanin pigments among Ipomoea species. The genetic basis for these transitions was examined using transgenics and gene expression assays. Using a literature survey to estimate the expected spectrum of mutation types capable of producing red flowers, we evaluated whether the observed distribution of mutation types differed from expectation. In these species, red floral pigmentation appears to be caused by the disruption of flux through the anthocyanin pathway at the same position. Results implicate tissue-specific regulatory changes in the same gene, which suggests the possibility that flower color evolved independently via the same genetic mechanism. Although multiple molecular mechanisms are capable of producing red flowers, we found a deviation between the distributions of observed and expected mutation types responsible for these evolutionary transitions. Regulatory mutations thus appear to be preferentially targeted during evolutionary change between species. We discuss possible explanations for this apparent bias.

Divergent selection drives genetic differentiation in an R2R3-MYB transcription factor that contributes to incipient speciation in Mimulus aurantiacus.

Identifying the molecular genetic basis of traits contributing to speciation is of crucial importance for understanding the ecological and evolutionary mechanisms that generate biodiversity. Despite several examples describing putative “speciation genes,” it is often uncertain to what extent these genetic changes have contributed to gene flow reductions in nature. Therefore, considerable interest lies in characterizing the molecular basis of traits that actively confer reproductive isolation during the early stages of speciation, as these loci can be attributed directly to the process of divergence. In Southern California, two ecotypes of Mimulus aurantiacus are parapatric and differ primarily in flower color, with an anthocyanic, red-flowered morph in the west and an anthocyanin-lacking, yellow-flowered morph in the east. Evidence suggests that the genetic changes responsible for this shift in flower color have been essential for divergence and have become fixed in natural populations of each ecotype due to almost complete differences in pollinator preference. In this study, we demonstrate that a cis-regulatory mutation in an R2R3-MYB transcription factor results in differential regulation of enzymes in the anthocyanin biosynthetic pathway and is the major contributor to differences in floral pigmentation. In addition, molecular population genetic data show that, despite gene flow at neutral loci, divergent selection has driven the fixation of alternate alleles at this gene between ecotypes. Therefore, by identifying the genetic basis underlying ecologically based divergent selection in flower color between these ecotypes, we have revealed the ecological and functional mechanisms involved in the evolution of pre-mating isolation at the early stages of incipient speciation.

Flower color as a model system for studies of plant evo-devo

Even though pigmentation traits have had substantial impacts on the field of animal evolutionary developmental biology, they have played only relatively minor roles in plant evo-devo. This is surprising given the often direct connection between flower color and fitness variation mediated through the effects of pollinators. At the same time, ecological and evolutionary genetic studies have utilized the molecular resources available for the anthocyanin pathway to generate several examples of the molecular basis of putatively adaptive transitions in flower color. Despite this opportunity to synthesize experimental approaches in ecology, evolution, and developmental biology, the investigation of many fundamental questions in evo-devo using this powerful model is only at its earliest stages. For example, a long-standing question is whether predictable genetic changes accompany the repeated evolution of a trait. Due to the conserved nature of the biochemical and regulatory control of anthocyanin biosynthesis, it has become possible to determine whether, and under what circumstances, different types of mutations responsible for flower color variation are preferentially targeted by natural selection. In addition, because plants use anthocyanin and related compounds in vegetative tissue for other important physiological functions, the identification of naturally occurring transitions from unpigmented to pigmented flowers provides the opportunity to examine the mechanisms by which regulatory networks are co-opted into new developmental domains. Here, we review what is known about the ecological and molecular basis of anthocyanic flower color transitions in natural systems, focusing on the evolutionary and developmental features involved. In doing so, we provide suggestions for future work on this trait and suggest that there is still much to be learned from the evolutionary development of flower color transitions in nature.

Strong premating reproductive isolation drives incipient speciation in Mimulus aurantiacus

Determining which forms of reproductive isolation have the biggest impact on the process of divergence is a major goal of speciation research. These barriers are often divided into those that affect the potential for hybridization (premating isolation), and those that occur after mating (postmating isolation), and much debate has surrounded the relative importance of these categories. Within the species Mimulus aurantiacus, red‐ and yellow‐flowered ecotypes occur in the southwest corner of California, and a hybrid zone occurs where their ranges overlap. We show that premating barriers are exclusively responsible for isolation in this system, with both ecogeographic and pollinator isolation contributing significantly to total isolation. Postmating forms of reproductive isolation have little or no impact on gene flow, indicating that hybrids likely contribute to introgression at neutral loci. Analysis of molecular variation across thousands of restriction‐site associated DNA sequencing (RAD‐seq) markers reveals that the genomes of these taxa are largely undifferentiated. However, structure analysis shows that these taxa are distinguishable genetically, likely due to the impact of loci underlying differentiated adaptive phenotypes. These data exhibit the power of divergent natural selection to maintain highly differentiated phenotypes in the face of gene flow during the early stages of speciation.

Introgressive hybridization facilitates adaptive divergence in a recent radiation of monkeyflowers

A primary goal in evolutionary biology is to identify the historical events that have facilitated the origin and spread of adaptations. When these adaptations also lead to reproductive isolation, we can learn about the evolutionary mechanisms contributing to speciation. We reveal the complex history of the gene MaMyb2 in shaping flower colour divergence within a recent radiation of monkeyflowers. In the Mimulus aurantiacus species complex, red-flowered M. a. ssp. puniceus and yellow-flowered M. a. ssp. australis are partially isolated because of differences in pollinator preferences. Phylogenetic analyses based on genome-wide variation across the complex suggest two origins of red flowers from a yellow-flowered ancestor: one in M. a. ssp. puniceus and one in M. a. ssp. flemingii. However, in both cases, red flowers are caused by cis-regulatory mutations in the gene MaMyb2. Although this could be due to distinct mutations in each lineage, we show that the red allele in M. a. ssp. puniceus did not evolve de novo or exist as standing variation in its yellow-flowered ancestor. Rather, our results suggest that a single red MaMyb2 allele evolved during the radiation of M. aurantiacus that was subsequently transferred to the yellow-flowered ancestor of M. a. ssp. puniceus via introgressive hybridization. Because gene flow is still possible among taxa, we conclude that introgressive hybridization can be a potent driver of adaptation at the early stages of divergence that can contribute to the origins of biodiversity.

The Genetic Basis of a Rare Flower Color Polymorphism in Mimulus lewisii Provides Insight into the Repeatability of Evolution

A long-standing question in evolutionary biology asks whether the genetic changes contributing to phenotypic evolution are predictable. Here, we identify a genetic change associated with segregating variation in flower color within a population of Mimulus lewisii. To determine whether these types of changes are predictable, we combined this information with data from other species to investigate whether the spectrum of mutations affecting flower color transitions differs based on the evolutionary time-scale since divergence. We used classic genetic techniques, along with gene expression and population genetic approaches, to identify the putative, loss-of-function mutation that generates rare, white flowers instead of the common, pink color in M. lewisii. We found that a frameshift mutation in an anthocyanin pathway gene is responsible for the white-flowered polymorphism found in this population of M. lewisii. Comparison of our results with data from other species reveals a broader spectrum of flower color mutations segregating within populations relative to those that fix between populations. These results suggest that the genetic basis of fixed differences in flower color may be predictable, but that for segregating variation is not.

Geographic cline analysis as a tool for studying genome‐wide variation: a case study of pollinator‐mediated divergence in a monkeyflower

A major goal of speciation research is to reveal the genomic signatures that accompany the speciation process. Genome scans are routinely used to explore genome‐wide variation and identify highly differentiated loci that may contribute to ecological divergence, but they do not incorporate spatial, phenotypic or environmental data that might enhance outlier detection. Geographic cline analysis provides a potential framework for integrating diverse forms of data in a spatially explicit framework, but has not been used to study genome‐wide patterns of divergence. Aided by a first‐draft genome assembly, we combined an FCT scan and geographic cline analysis to characterize patterns of genome‐wide divergence between divergent pollination ecotypes of Mimulus aurantiacus. FCT analysis of 58 872 SNPs generated via RAD‐seq revealed little ecotypic differentiation (mean FCT = 0.041), although a small number of loci were moderately‐to‐highly diverged. Consistent with our previous results from the gene MaMyb2, which contributes to differences in flower colour, 130 loci have cline shapes that recapitulate the spatial pattern of trait divergence, suggesting that they may reside in or near the genomic regions that contribute to pollinator isolation. In the narrow hybrid zone between the ecotypes, extensive admixture among individuals and low linkage disequilibrium between markers indicate that most outlier loci are scattered throughout the genome, rather than being restricted to one or a few divergent regions. In addition to revealing the genomic consequences of ecological divergence in this system, we discuss how geographic cline analysis is a powerful but under‐utilized framework for studying genome‐wide patterns of divergence.

Genomic studies on the nature of species: adaptation and speciation in Mimulus

Evolutionary biology is in an exciting era, in which powerful genomic tools make the answers accessible to long‐standing questions about variation, adaptation and speciation. The availability of a suite of genomic resources, a shared knowledge base and a long history of study have made the phenotypically diverse plant genus Mimulus an important system for understanding ecological and evolutionary processes. An international Mimulus Research Meeting was held at Duke University in June 2014 to discuss developments in ecological and evolutionary genetic studies in Mimulus. Here, we report major recent discoveries presented at the meeting that use genomic approaches to advance our understanding of three major themes: the parallel genetic basis of adaptation; the ecological genomics of speciation; and the evolutionary significance of structural genetic variation. We also suggest future research directions for studies of Mimulus and highlight challenges faced when developing new ecological and evolutionary model systems.