New Phytologist

Most plants form mutualistic symbioses with soil fungi, including arbuscular mycorrhizal (AM) fungi. These fungi usually transfer soil nutrients to plants and assimilate carbon from host plant photosynthesis. Recently, Mucoromycotina fine root endophytes (MFRE) were identified as nutritionally mutualistic and widespread fungal symbionts of plants, establishing MFRE as a new class of mycorrhizal fungi. However, the regulatory mechanisms for MFRE symbioses are completely unknown. Other symbionts, like AM fungi, use the Common Symbiotic Signalling Pathway (CSSP) to establish symbiosis. To explore whether MFRE interactions also involve this pathway, we cultured MFRE with CSSP mutants of Medicago truncatula which show impaired AM symbioses and tracked carbon and nutrient transfers using isotope tracers. Results show no differences in root colonization or nutrient exchange, suggesting MFRE symbioses are regulated by different molecular mechanisms. This finding highlights the unique nature of MFRE symbiosis, broadening our understanding of diverse fungal symbioses and their evolutionary significance.

Diatoms synthesize silica cell walls (frustules) with genetically encoded morphologies, ranging from nanopatterns to micropatterns, that far exceed current synthetic chemistry. Silaffins, a family of phosphoproteins undergoing complex post-translational modifications, have been isolated from frustules and shown to facilitate and regulate biosilica formation in vitro with long-chain polyamines. However, their particular role in frustule morphogenesis and functionality remains unclear. In this study, functions of two representative silaffins, TpSil1 and TpSil3, were investigated in the model organism Thalassiosira pseudonana using gene overexpression and CRISPR/Cas9-mediated knockout approaches. Due to high sequence homology, TpSil2 was concurrently disrupted in TpSil1 knockout strains, while the homozygous knockout of TpSil3 proved to be lethal. Quantitative morphological analysis revealed distinct yet complementary roles: TpSil3 governs both microscale overall size and mesoscale features, including macropore (fultoportula) density and mesopore (cribrum pore) pattern, whereas TpSil1/2 exclusively contribute to macropore morphogenesis and mesopore density. Overexpression of silaffins increased silica deposition, while knockouts exhibited reduced silicification but enhanced cell growth and photosynthetic efficiency. Furthermore, these genetic modifications significantly influenced the physicochemical and optical properties of bulk frustules, potentially enhancing the hemostatic, catalytic and photonic performances, thereby positioning them as versatile candidates for a wide range of biotechnological and industrial applications. Collectively, our findings elucidate the distinct roles of TpSil1/2 and TpSil3 in diatom physiology and frustule morphology, highlighting a promising pathway for engineering nanostructured silica materials with tailored properties through synthetic biology.

Global nitrogen (N) deposition has fundamentally reshaped plant nutrient acquisition by altering mechanistic trade-offs between root exploration-exploitation strategies and mycorrhizal symbiosis. Through a meta-analysis of 135 studies spanning 153 sites, we demonstrate that N deposition stimulates root exploration (+84.6% root length density) and exploitation (+13.2% nitrogen content and +8.5% root biomass) while suppressing mycorrhizal dependence (-32.8% biomass, -18.73% colonized root length, -7.55% hyphal length), indicating a systemic shift toward root-autonomous nutrient acquisition. Divergent responses emerged between plant lifeforms: woody plants prioritize exploitation (+13.2% nitrogen content) over exploration capacity (-7.9% root length density), whereas herbaceous species exhibit synergistic enhancement of both strategies (+88% root length density, +13.2% nitrogen content and +71.3% root biomass). In contrast to N-only effects, Combined nitrogen and phosphorus additions reversed mycorrhizal suppression (+41.9% biomass), highlighting their persistent role in phosphorus acquisition under elevated nutrient conditions. The root economics space framework demonstrates predictive power, with plants possessing lower specific root length (SRL) and higher nitrogen content exhibiting stronger positive responses to N deposition, while those possessing higher SRL and lower nitrogen content showed reduced investment in root systems. These trait-mediated thresholds in carbon-nutrient tradeoffs refine our capacity to model belowground ecological responses to anthropogenic nitrogen perturbation, establishing a mechanistic basis for projecting ecosystem trajectories under global change.

O_LIPhytochromes are photoreceptors enabling plants to respond to various light conditions. Independent gene duplication events resulted in small phytochrome gene families in mosses, ferns, and seed plants. This phytochrome diversity is hypothesised to be critical for sensing and adapting to different light conditions, but experimental evidence for this idea is lacking for mosses and ferns. C_LIO_LIThe model moss species Physcomitrium patens contains seven phytochromes grouped into three clades, PHY1/3, PHY2/4, and PHY5. Here, we used CRISPR/Cas9 generated single and higher order mutants to investigate their role in light-regulation of protonema and gametophore growth, protonema branching, and induction of gametophores. C_LIO_LIWe found both specific and partially overlapping roles for the three clades of moss phytochromes in regulating these responses in different light conditions, and we identified a mechanism for sensing simulated canopy shade different from the mechanism in seed plants. PHY1/3 clade phytochromes act as primary far-red light receptors, while PHY5 clade phytochromes are the primary red light receptors. PHY2/4 clade phytochromes have functions in both red and far-red light. C_LIO_LISimilar to seed plants, gene duplication events in the phytochrome lineage in mosses were followed by functional diversification into red and far-red light sensing phytochromes. C_LI

O_LIPlant functional traits vary across leaf ontogeny and phenology, yet most trait data are snapshots from narrow time windows that miss this temporal dimension. Leaf spectra are increasingly used with empirical models to predict traits, but whether such models accurately capture phenological variation remains unclear. C_LIO_LIWe monitored leaf traits and spectra weekly across a full growing season, generating 7,515 spectra from seven temperate species. Using partial least squares regression, we built three models --all-season and week-as-covariate models (both trained on full-phenology data), and a peak-season model -- and evaluated them alongside a widely used model against directly measured traits. C_LIO_LIFull-phenology models predicted LMA and equivalent water thickness (EWT) with high accuracy (R{superscript 2} > 0.85) and nitrogen with intermediate accuracy (R{superscript 2} = 0.64); carbon accuracy was low across all models (R{superscript 2} < 0.26), likely due to a small sample size. Peak-season trained models performed poorly when evaluated across the full season, often producing biologically unrealistic predictions. Traits and spectra varied significantly across phenological stages both within and among species. C_LIO_LIIgnoring phenological variation systematically biases trait estimates and ecological inference. Coupled with phenologically representative training data, spectra can capture the temporal dynamics of plant function, enabling novel research in ecology and evolution. C_LI

O_LIPollination is essential for plant reproduction, ecosystem resilience and human health. Yet, our capability to map pollination service delivery in real-time across large areas remains poor. Determining where and when flowers are pollinated is vital to mitigate widespread pollination deficits, increase plant health and yield, and support pollinator management. Hence, innovative approaches are urgently needed for establishing scalable predictive bioindicators of plant pollination status with the goal of achieving real-time landscape-scale monitoring. C_LIO_LIHere we present two parallel controlled pollination assays in which we characterise the post-pollination petal physiology of a world leading flowering crop, Brassica napus, using in-situ close-range hyperspectral reflectance and semi-untargeted metabolomics. C_LIO_LIThis multiomics approach coupled with supervised machine learning and biomarker detection reveals cryptic changes in the UV petal reflectance spectrum which are predictive of pollination status, representing a novel set of candidate pollination bioindicators ( polli-markers), and our high-resolution time series enables prediction of when this pollination event occurred. It also reveals an associated set of candidate metabolites, including flavonoids and senescence markers, shedding light on the functional pathways related to our polli-markers. C_LIO_LIThis study provides key insights into floral development, enabling a transformative step towards predicting, mapping and quantifying pollination service delivery at the landscape scale. C_LI

Phytochemistry is a core player in shaping plant-pollinator networks and pollination services. Yet, little is known about the dynamic evolution of phytochemical traits in response to limited pollinator access, especially concerning chemical diversity. We combined an evolutionary experiment manipulating pollinator access with predictive metabolomics to uncover evolutionary changes in phytochemical traits of Brassica rapa. Our results unveiled chemical changes in both leaf and flower chemistry. Moreover, plants under selection by limited pollinator access showed a decreased chemical richness and diversity and a modulated primary and specialised metabolism, which could be used to predict pollinator access with 88% accuracy. Chemical indices and metabolites responding to pollinator access were associated with variation in flowering time and performance of outcrossing flowers. Our findings provide key insights into the influence of pollinator access on plant chemistry and indicate a risk of pollinator decline and losses of chemical diversity for plant-pollinator network structure and ecosystem dynamics.

Quantifying the variation in plant traits reveals the trade-offs involved in plant ecological strategies and is fundamental to understanding underlying plant fitness mechanisms. Thus, the ecological success of plant species in a certain habitat may depend on the coordinated performance of both leaves and roots. However, despite the growing interest in trait variation, it is still uncertain i) to what extent the leaf economics spectrum (LES) and root economics space (RES) hold across locally coexisting tree species and ii) whether leaf and fine-root traits are correlated. In a research arboretum, we simultaneously measured eight key traits in leaves and fine-roots on 270 individuals belonging to 90 tree species, encompassing both angiosperm and gymnosperm species. We find varied plant resource strategies associated with leaves and fine-roots for angiosperms and gymnosperms. We observe a clear LES for gymnosperms and a clear RES for angiosperms. Our results support the existence of a correlation between analogous leaf and fine-root traits across all species. However, varying trait coordination across clades indicates varying resource acquisition strategies above- and belowground, highlighting the need to consider large-scale phylogenetic relatedness to better understand plant fitness.

Annual and perennial life-history strategies have evolved repeatedly across angiosperms, yet the genomic and environmental underpinnings of these transitions remain poorly understood, particularly in grasses. We generated de novo transcriptomes from 82 accessions representing 22 Hordeum species, including barley, and identified 257 single-copy orthologs present in all accessions to infer a robust phylogeny of the genus. By combining phylogenetic network inference with ABBA-BABA tests, we detected four cases of interspecific hybridization, three coinciding with major long-distance dispersal events across continents. Comparative climatic niche analysis indicated annual Hordeum species inhabit environments with higher temperatures, greater interannual variability, and increased human disturbance, compared to perennials, although no consistent precipitation differences were observed. Using our phylogeny as a framework, we analyzed selection and gene expression to uncover genomic changes associated with life-history strategy while accounting for phylogenetic non-independence. Additionally, we analysed gene copy number variations associated with life-history strategy. These analyses yielded 174 candidate genes across diverse biological functions, suggesting the genetic architecture underlying life-history evolution is more complex than assumed. Candidate genes grouped into six major functional categories, the most prominent being signal transduction and development, including regulators of flowering, dormancy, and meristem activity, metabolic and biosynthetic processes related to carbon allocation and storage, and stress response and defense, reflecting the resilience of perennials compared to the accelerated growth strategies of annuals. Our study reconstructs the evolutionary history and climatic niche differentiation of Hordeum species and demonstrates that convergent life-history evolution is driven by multifaceted, functionally diverse genetic mechanisms.

While DNA has built the framework for molecular insights from museum collections, the utility of archival RNA remains largely unexplored. Likely a consequence of the known instability of RNA relative to DNA, this has effectively precluded the use of herbaria for transcriptomics. Here, we challenge the assumption that herbaria cannot be used for transcriptomics by assembling transcriptomes from RNA extracted from herbarium specimens. Through systematic comparison of transcriptomes from fresh-collected, silica-dried, and archival specimens, we demonstrate the suitability of herbarium-derived RNA for transcriptomics. We show the practical applicability of archival mRNA by functionally validating a plant immune receptor synthesized from a specimen collected in 1956. These results contradict the community consensus regarding archival RNA and open the door to subsequent transcriptomic explorations of rare and extinct plant species. Our findings highlight the importance of preserving and utilizing the diversity embedded within herbarium collections.

O_LIGrasses in the PACMAD clade independently colonized cold environments from warm-climate ancestors, but whether their molecular responses to freezing reflect shared evolutionary solutions or lineage-specific innovations remains unknown. We used comparative proteomics to test whether protein-level cold responses show stronger cross-species conservation than previously observed at the transcript level. C_LIO_LIWe quantified seasonal rhizome proteomes (winter vs summer) from five PACMAD species grown in a common garden exposed to sustained sub-zero temperatures, identified differentially abundant proteins, and compared fold-change magnitudes across species using orthogroup-based correlation analyses. We further examined LEA3 protein structure through hydropathy profiling and motif analysis. C_LIO_LIShared cold-responsive proteins showed higher cross-species fold-change correlation ({rho} = 0.80) than background proteins ({rho} = 0.45), despite greater divergence in baseline abundance. LEA3 was the only ortholog elevated across all five species. Cold-tolerant species contained more tandem 11-mer repeats than the cold-sensitive maize, and two species accumulated multiple LEA3 paralogs, increasing total LEA3 abundance. C_LIO_LIIndependent evolution of freezing tolerance in PACMAD grasses is governed by evolutionary constraints on protein-level response magnitude, reflecting the retention of an ancestral protective capacity. Structural divergence of LEA3 in maize suggests that transcriptional induction alone does not ensure freezing tolerance; functional protection likely requires intact motif architecture. C_LI

O_LIThe importance of functional trait evolution and genome size on plant speciation are well established, but their interactive effects remain untested in a single comparative macroevolutionary framework. C_LIO_LIWe integrated phylogenetic, trait and genome size data for palms (Arecaceae) - a large pantropical family (>2600 species) with 167-fold variation in trait and 60-fold variation in genome size. We used structural equation modelling to test three key hypotheses: trait evolution promotes speciation (H1: trait flexibility hypothesis), and, speciation and trait evolution rates are constrained by allometry (H2: allometric constraint hypothesis) and genome size (H3: large genome constraint hypothesis). C_LIO_LIWe detected seven major speciation rate shifts during the ca. 110-million-year history of palms. Tip-derived speciation rates increased with faster evolution in leaves and plant height, supporting H1, whereas correlated evolution between trait evolution rates indirectly influenced speciation, supporting H2. Large genomes decreased plant height and stem diameter evolutionary rates, but increased leaf size evolution and speciation rates, thus partly supporting H3. C_LIO_LIOur findings illustrate how the complex interplay between genome size, allometry and trait evolvability affect speciation, emphasizing the importance of holistic approaches in macroevolution. Furthermore, our results point to potential general mechanisms driving speciation rates throughout the plant Tree of Life. C_LI

Background and AimsWhile genome size limits the minimum sizes and maximum numbers of cells that can be packed into a given leaf volume, mature cell sizes can be substantially larger than their meristematic precursors and vary in response to abiotic conditions. Mangroves are iconic examples of how abiotic conditions can influence the evolution of plant phenotypes. MethodsHere, we examined the coordination between genome size, leaf cell sizes, and cell packing densities, and leaf size in 13 mangrove species across four sites. Four of these species occurred at more than one site, allowing us to test the effect of climate on leaf anatomy. ResultsWe found that genome sizes of mangroves were very small compared to other angiosperms, and, like other angiosperms, mangrove cells were always larger than the minimum size defined by genome size. Increasing mean annual temperature of a growth site led to higher packing densities of veins (Dv) and stomata (Ds) and smaller epidermal cells but had no effect on stomatal size. Contrary to other angiosperms, mangroves exhibited (1) a negative relationship between guard cell size and genome size; (2) epidermal cells that were smaller than stomata, and (3) coordination between Dv and Ds that was not mediated by epidermal cell size. Furthermore, mangrove epidermal cell sizes and packing densities covaried with leaf size. ConclusionsWhile mangroves exhibited coordination between veins and stomata and attained a maximum theoretical stomatal conductance similar to other angiosperms, the tissue-level tradeoffs underlying these similar relationships across species and environments was markedly different, perhaps indicative of the unique structural and physiological adaptations of mangroves to their stressful environments.

Although distributed globally, plants possessing the succulent syndrome are thought to have evolved to adapt to arid climates, because they possess modifications that increase their water use efficiency. Here we study the evolution and the ecological nature of the succulent CAM syndrome at a global scale by analyzing the climatic niches of succulents within the Caryophyllales, testing the hypothesis of a climatic niche specialization by comparing them with their non-succulent, non-arid adapted relatives. We assembled and carefully curated a worldwide dataset of 5447 species in 28 families, and analyzed the current and evolutionary trajectories of climatic niches with an array of statistical methods including ecological niche modeling, phylogenetic regression and divergence dates estimation. Our results confirm the Core Caryophyllales tend to inhabit drylands probably since their origin in the Early Cretaceous. However, the succulent syndrome appeared later with some lineages diversifying profusely afterwards. The climatic niche of succulents is not differentiated from their non-succulent relatives, but narrower, and contained within the non-succulents, showing no relationship with extreme conditions such as high aridity or temperatures. Our results support alternative interpretations of the origin of the CAM syndrome and the ecological significance of succulence, as well as the prolific radiation of richest lineages. HighlightsThe climatic niche occupied by succulent CAM plants is not different from their non-succulent relatives. Estimated dates and character reconstruction suggest CO2 scarcity as the evolutionary pressure under these plants originated.

Liverworts can clonally propagate by producing compact shoot structures called gemmae, which develop within basket-like structures known as gemma cups. It was previously reported in Marchantia nepalensis that carbon availability promotes gemma cup formation. However, the mechanisms by which carbon availability controls this process remains largely unexplored. To address this knowledge gap, we investigated how carbon promotes gemma cup formation using Marchantia polymorpha as a model species. Through a series of pharmacological and genetic experiments, we found that carbon availability promotes gemma cup formation by inducing the cytokinin pathway, thereby increasing the expression of MpGCAM1 and MpSTG, which encode two transcription factors involved in forming the basal floor of gemma cups. Indeed, our data show that cytokinins accumulate in marchantia thallus in response to sucrose and to high light treatments. In addition, constitutive induction of cytokinin signalling could overcome the repressive effect of low sucrose on gemma cup formation, whereas suppression of this hormonal pathway led to inhibition of sucrose-induced gemma cup formation. Furthermore, our results indicate that sucrose can induce gemma cup formation independently of KAI2A and MAX2, two molecular components of karrikin signalling known to control this developmental process by inducing cytokinin synthesis. Interestingly, in flowering plants, carbon availability also promotes cytokinin accumulation to induce axillary bud outgrowth, a process involving the transcription factors AtRAX and AtLOF1, the Arabidopsis thaliana orthologues of MpGCAM1 and MpSTG, respectively. Collectively, these observations indicate that the interactions between carbon and cytokinins are critical for the developmental plasticity of land plants in response to their environment.

O_LIAmphicarpy is an unusual trait where two fruit types develop: one above and the other below ground. This trait is not found in conventional model species, therefore, its development and molecular genetics remain under-studied. Here, we establish Cardamine chenopodiifolia as an emerging experimental system to study amphicarpy. C_LIO_LIWe characterized the development of C. chenopodiifolia, focusing on differences in morphology and cell wall histochemistry between above- and below-ground fruit. We generated a reference transcriptome using PacBio full-length transcript sequencing (IsoSeq) and used a combination of short and long read sequencing to analyse differential gene expression between above- and below-ground fruit valves. C_LIO_LIC. chenopodiifolia has two contrasting modes of seed dispersal. The main shoot fails to bolt and initiates floral primordia that bury underground where they self-pollinate and set seed. By contrast, axillary shoots bolt to position flowers and exploding seed pods above ground. Morphological differences between aerial explosive fruit and subterranean non-explosive fruit were reflected in a large number of differentially regulated genes involved in photosynthesis, secondary cell wall formation and defence responses. C_LIO_LITools established in C. chenopodiifolia, such as a reference transcriptome, draft genome assembly and stable plant transformation, pave the way to explore under-studied traits and discover new biological mechanisms. C_LI

Beneficial root-associated microbes can enhance plant resilience by complementing aspects of host immunity. The fungal root endophyte Serendipita indica (Si) is known to promote plant growth and confer broad stress tolerance. To assess how natural host genetic variation influences Si-mediated protection, we screened 47 Arabidopsis thaliana accessions for susceptibility to the fungal pathogen Bipolaris sorokiniana (Bs) with and without Si colonization. All accessions benefited from Si, indicating that endophyte-mediated disease mitigation occurs broadly across diverse host genotypes. A focused comparison of two genetically and geographically proximate Swedish accessions, T510 and T530, which displayed the most divergent protection scores, revealed substantial differences in Bs susceptibility. Transcriptome profiling under bi- and tripartite colonization showed conserved defense responses in both accessions. Bs infection downregulated growth- and development-related genes, consistent with a growth-immunity trade-off, with T530 exhibiting higher Bs colonization and a stronger transcriptional response than T510. Co-colonization with Si effectively suppressed pathogen growth and disease symptoms in both accessions. Comparative genomic and transcriptomic analyses identified four immune receptor genes, including the TIR-NLR ISI, present in T510 but absent in T530. An isi T-DNA insertion mutant phenocopied the heightened Bs susceptibility of T530, confirming that ISI contributes to root immunity, while Si-mediated protection remained intact despite increased pathogen susceptibility. Together, these findings demonstrate that fungal endophytes can mitigate the functional consequences of natural immune variation and enhance the resilience of genetically diverse plant populations. HighlightsO_LIS. indica confers broad protection against B. sorokiniana largely independent of host genotype or pathogen susceptibility. C_LIO_LIThe TIR-NLR immune receptor ISI contributes to root immunity but is not essential for S. indica-mediated protection. C_LIO_LIBeneficial endophytes can mitigate natural immune variation effects and support overall plant health. C_LI

O_LICrassulacean acid metabolism (CAM) photosynthesis has independently evolved many times in arid-adapted plant lineages. Cistanthe longiscapa (Montiaceae), a desert mass-blooming annual, can upregulate CAM facultatively upon stress such as drought. Few studies, however, consider life history stages when measuring CAM activity or its facultative onset. C_LIO_LITo test the effect of drought and flowering on photosynthetic activity, we assayed Cistanthe individuals in fully-watered and drought conditions, as well as fully-watered individuals at pre-flowering and flowering life stages. We assembled and annotated a chromosome-scale genome of C. longiscapa and compared it with the genome of Portulaca amilis and analyzed differential gene expression. C_LIO_LIResults show significantly upregulated CAM in drought conditions as compared to fully-watered conditions; furthermore, flowering individuals showed slightly higher CAM activity as compared to pre-flowering plants, even when fully-watered. Differential gene expression analyses provide preliminary support for the possible co-regulation of CAM expression and reproduction. C_LIO_LIWe emphasize the potentially missed significance of life history in the CAM literature, and consider how the CAM biochemical module could become co-opted into other plant behaviors and responses, such as the shift to reproduction or flowering in annuals. C_LI

BackgroundMost land plants depend on the ancestral arbuscular mycorrhizal (AM) symbiosis for phosphorus (P) acquisition. However, several plant lineages have independently lost this symbiosis, raising fundamental questions about how these non-mycorrhizal plants meet their nutritional requirements without this crucial partnership. ResultsComparative genomic analyses confirmed that Cyperaceae, Caryophyllaceae, and Brassicaceae lack genes essential for AM symbiosis, indicating that these lineages independently abandoned this association 90-122 million years ago. Field surveys of 42 wild populations across seven sites revealed that while non-mycorrhizal plants generally maintain shoot P levels comparable to those in AM neighbors, lower shoot P levels can be observed in low P soils. To identify fungal taxa potentially associated with P nutrition in non-mycorrhizal plants, we applied a machine-learning approach to predict plant P-accumulation from root microbiome composition. The model explained substantial variance in plant P-accumulation (57-69%), and identified 85 fungal taxa as key predictors of shoot P-accumulation, predominantly belonging to the Helotiales (28%) and Pleosporales (23%) orders. Experimental validation of two phylogenetically distant Helotiales lineages (Tetracladium maxilliforme OTU29 and Helotiales sp. OTU7), using isotopic tracing, demonstrated their capacity to enhance plant growth and transfer P (and N) to their native non-mycorrhizal hosts under P-limiting conditions. ConclusionsOur findings suggest that non-mycorrhizal plants engage in nutritional partnerships with diverse Helotiales lineages that could collectively contribute to their mineral nutrition. However, given the widespread distribution of these Helotiales fungi, including in roots of AM plants, they may play a broader role in plant nutrition, i.e. also in mycorrhizal hosts. This study provides proof of concept for a novel framework integrating machine-learning predictions with experimental validation to identify functionally important microbial partnerships in natural plant communities.

The process of reinforcement, whereby selection favors the evolution of increased reproductive trait divergence to reduce costly hybridization between species, has been well documented in nature, yet we know very little about how this process evolves at the molecular level. In this study, we combine functional characterization and genetic association tests to identify the mutational basis of reinforcement in the Texas wildflower Phlox drummondii. P. drummondii evolved from light to dark flower color intensity by selection to stop hybridization with the closely related species P. cuspidata, and previous research suggests differential expression of a R2R3-Myb transcription factor underlies this phenotypic transition. Using gene-silencing experiments, we demonstrate expression of this transcription factor does control variation in flower color intensity. We then apply association mapping across a large genomic region flanking the R2R3-Myb gene and identified a point mutation within the genes promoter that is highly associated with flower color intensity in nature. Alleles at this mutation site match the expected patterns of dominance, create variation in predicted cis-regulatory motifs within the R2R3-Myb proximal promoter, and occur in the direction of evolution predicted for flower color variation in this system. By identifying the mutational basis of reinforcement in this system we demonstrate that, as predicted by theory, reproductive isolation can evolve despite gene flow through a very simple genetic basis.