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.

CYCLOIDEA paralogs function partially redundantly to specify dorsal flower development in Mimulus lewisii.

PREMISE: Duplicated genes (paralogs) are abundant in plant genomes, and their retention may influence the function of genetic programs and contribute to evolutionary novelty. How gene duplication affects genetic modules and what forces contribute to paralog retention are outstanding questions. The CYCLOIDEA(CYC)-dependent flower symmetry program is a model for understanding the evolution of gene duplication, providing multiple examples of paralog partitioning and novelty. However, a novel CYC gene lineage duplication event near the origin of higher core Lamiales (HCL) has received little attention. METHODS: To understand the evolutionary fate of duplicated HCL CYC2 genes, we determined the effects on flower symmetry by suppressing MlCYC2A and MlCYC2B expression using RNA interference (RNAi). We determined the phenotypic effects on flower symmetry in single- and double-silenced backgrounds and coupled our functional analyses with expression surveys of MlCYC2A, MlCYC2B, and a putative downstream RADIALIS (MlRAD5) ortholog. RESULTS: MlCYC2A and MlCYC2B jointly contribute to bilateral flower symmetry. MlCYC2B exhibits a clear dorsal flower identity function and may additionally function in carpel development. MlCYC2A functions in establishing dorsal petal shape. Further, our results suggest an MlCYC2A-MlCYC2B regulatory interaction, which may affect pathway homeostasis. CONCLUSIONS: Our results suggest that CYC paralogs specific to higher core Lamiales may be selectively retained for their joint contribution to bilateral flower symmetry, similar to the independently derived CYC paralogs in the Lamiales model for bilateral flower symmetry research, Antirrhinum majus (snapdragon).

Nectary size is a pollination syndrome trait in Penstemon.

Evolution of complex phenotypes depends on the adaptive importance of individual traits, and the developmental changes required to modify traits. Floral syndromes are complex adaptations to pollinators that include color, nectar, and shape variation. Hummingbird-adapted flowers have evolved a remarkable number of times from bee-adapted ancestors in Penstemon, and previous work demonstrates that color over shape better distinguishes bee from hummingbird syndromes. Here, we examined the relative importance of nectar volume and nectary development in defining Penstemon pollination syndromes. We tested the evolutionary association of nectar volume and nectary area with pollination syndrome across 19 Penstemon species. In selected species, we assessed cellular-level processes shaping nectary size. Within a segregating population from an intersyndrome cross, we assessed trait correlations between nectar volume, nectary area, and the size of stamens on which nectaries develop. Nectar volume and nectary area displayed an evolutionary association with pollination syndrome. These traits were correlated within a genetic cross, suggesting a mechanistic link. Nectary area evolution involves parallel processes of cell expansion and proliferation. Our results demonstrate that changes to nectary patterning are an important contributor to pollination syndrome diversity and provide further evidence that repeated origins of hummingbird adaptation involve parallel developmental processes in Penstemon.

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.

Accessibility, constraint, and repetition in adaptive floral evolution.

Adaptive phenotypic evolution is shaped by natural selection on multiple organismal traits as well as by genetic correlations among traits. Genetic correlations can arise through pleiotropy and can bias the production of phenotypic variation to certain combinations of traits. This phenomenon is referred to as developmental bias or constraint. Developmental bias may accelerate or constrain phenotypic evolution, depending on whether selection acts parallel or in opposition to genetic correlations among traits. We discuss examples from floral evolution where genetic correlations among floral traits contribute to rapid, coordinated evolution in multiple floral organ phenotypes and suggest future research directions that will explore the relationship between the genetic basis of adaptation and the pre-existing structure of genetic correlations. On the other hand, natural selection may act perpendicular to a strong genetic correlation, for example when two traits are encoded by a subset of the same genes and natural selection favors change in one trait and stability in the second trait. In such cases, adaptation is constrained by the availability of genetic variation that can influence the focal trait with minimal pleiotropic effects. Examples from plant diversification suggest that the origin of certain adaptations depends on the prior evolution of a gene copy with reduced pleiotropic effects, generated through the process of gene duplication followed by subfunctionalization or neofunctionalization. A history of gene duplication in some developmental pathways appears to have allowed particular flowering plant linages to have repeatedly evolved adaptations that might otherwise have been developmentally constrained.

Repeated and diverse losses of corolla bilateral symmetry in the Lamiaceae.

Background and Aims: Independent evolution of derived complex characters provides a unique opportunity to assess whether and how similar genetic changes correlate with morphological convergence. Bilaterally symmetrical corollas have evolved multiple times independently from radially symmetrical ancestors and likely represent adaptations to attract specific pollinators. On the other hand, losses of bilateral corolla symmetry have occurred sporadically in various groups, due to either modification of bilaterally symmetrical corollas in late development or early establishment of radial symmetry. Methods: This study integrated phylogenetic, scanning electron microscopy (SEM)-based morphological, and gene expression approaches to assess the possible mechanisms underlying independent evolutionary losses of corolla bilateral symmetry. Key Results: This work compared three species of Lamiaceae having radially symmetrical mature corollas with a representative sister taxon having bilaterally symmetrical corollas and found that each reaches radial symmetry in a different way. Higher core Lamiales share a common duplication in the CYCLOIDEA (CYC ) 2 gene lineage and show conserved and asymmetrical expression of CYC2 clade and RAD genes along the adaxial-abaxial floral axis in species having bilateral corolla symmetry. In Lycopus americanus , the development and expression pattern of La-CYC2A and La-CYC2B are similar to those of their bilaterally symmetrical relatives, whereas the loss of La-RAD expression correlates with a late switch to radial corolla symmetry. In Mentha longifolia , late radial symmetry may be explained by the loss of Ml-CYC2A , and by altered expression of two Ml-CYC2B and Ml-RAD genes . Finally, expanded expression of Cc-CYC2A and Cc-RAD strongly correlates with the early development of radially symmetrical corollas in Callicarpa cathayana . Conclusions: Repeated losses of mature corolla bilateral symmetry in Lamiaceae are not uncommon, and may be achieved by distinct mechanisms and various changes to symmetry genes, including the loss of a CYC2 clade gene from the genome, and/or contraction, expansion or alteration of CYC2 clade and RAD -like gene expression.

Non-equilibrium dynamics and floral trait interactions shape extant angiosperm diversity.

Why are some traits and trait combinations exceptionally common across the tree of life, whereas others are vanishingly rare? The distribution of trait diversity across a clade at any time depends on the ancestral state of the clade, the rate at which new phenotypes evolve, the differences in speciation and extinction rates across lineages, and whether an equilibrium has been reached. Here we examine the role of transition rates, differential diversification (speciation minus extinction) and non-equilibrium dynamics on the evolutionary history of angiosperms, a clade well known for the abundance of some trait combinations and the rarity of others. Our analysis reveals that three character states (corolla present, bilateral symmetry, reduced stamen number) act synergistically as a key innovation, doubling diversification rates for lineages in which this combination occurs. However, this combination is currently less common than predicted at equilibrium because the individual characters evolve infrequently. Simulations suggest that angiosperms will remain far from the equilibrium frequencies of character states well into the future. Such non-equilibrium dynamics may be common when major innovations evolve rarely, allowing lineages with ancestral forms to persist, and even outnumber those with diversification-enhancing states, for tens of millions of years.

Paralogous SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes differentially regulate leaf initiation and reproductive phase change in petunia.

MAIN CONCLUSION: Duplicated petunia clade-VI SPL genes differentially promote the timing of inflorescence and flower development, and leaf initiation rate. The timing of plant reproduction relative to favorable environmental conditions is a critical component of plant fitness, and is often associated with variation in plant architecture and habit. Recent studies have shown that overexpression of the microRNA miR156 in distantly related annual species results in plants with perennial characteristics, including late flowering, weak apical dominance, and abundant leaf production. These phenotypes are largely mediated through the negative regulation of a subset of genes belonging to the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) family of transcription factors. In order to determine how and to what extent paralogous SPL genes have partitioned their roles in plant growth and development, we functionally characterized petunia clade-VI SPL genes under different environmental conditions. Our results demonstrate that PhSBP1and PhSBP2 differentially promote discrete stages of the reproductive transition, and that PhSBP1, and possibly PhCNR, accelerates leaf initiation rate. In contrast to the closest homologs in annual Arabidopsis thaliana and Mimulus guttatus, PhSBP1 and PhSBP2 transcription is not mediated by the gibberellic acid pathway, but is positively correlated with photoperiod and developmental age. The developmental functions of clade-VI SPL genes have, thus, evolved following both gene duplication and speciation within the core eudicots, likely through differential regulation and incomplete sub-functionalization.

Multiplexed shotgun genotyping resolves species relationships within the North American genus Penstemon.

PREMISE OF THE STUDY: Evolutionary radiations provide opportunities to examine large-scale patterns in diversification and character evolution, yet are often recalcitrant to phylogenetic resolution due to rapid speciation events. The plant genus Penstemon has been difficult to resolve using Sanger sequence-based markers, leading to the hypothesis that it represents a recent North American radiation. The current study demonstrates the utility of multiplexed shotgun genotyping (MSG), a style of restriction site-associated DNA sequencing (RADseq), to infer phylogenetic relationships within a subset of species in this genus and provide insight into evolutionary patterns. METHODS: We sampled genomic DNA, primarily from herbarium material, and subjected it to MSG library preparation and Illumina sequencing. The resultant sequencing reads were clustered into homologous loci, aligned, and concatenated into data matrices that differed according to clustering similarity and amount of missing data. We performed phylogenetic analyses on these matrices using maximum likelihood (RAxML) and a species tree approach (SVDquartets). KEY RESULTS: MSG data provide a highly resolved estimate of species relationships within Penstemon. While most species relationships were highly supported, the position of certain taxa remains ambiguous, suggesting that increased taxonomic sampling or additional methodologies may be required. The data confirm that evolutionary shifts from hymenopteran- to hummingbird-adapted flowers have occurred independently many times. CONCLUSIONS: This study demonstrates that phylogenomic approaches yielding thousands of variable sites can greatly improve species-level resolution of recent and rapid radiations. Similar to other studies, we found that less conservative similarity and missing data thresholds resulted in more highly supported topologies.

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.

Evolutionary analysis of the MIXTA gene family highlights potential targets for the study of cellular differentiation.

Differentiated epidermal cells such as trichomes and conical cells perform numerous essential functions in plant biology and are important for our understanding of developmental patterning and cell shape regulation. Many are also commercially significant, such as cotton fibers and trichomes that secrete pharmaceutically useful or herbivore-deterring compounds. Here, we focus on the phylogeny and evolution of the subgroup 9 R2R3 MYB gene transcription factors, which include the MIXTA gene, and that are important for the specification and regulation of plant cellular differentiation. We have sequenced 49 subgroup 9 R2R3 MYB genes from key experimental taxa and combined these sequences with those identified by an exhaustive bioinformatic search, to compile a data set of 223 subgroup 9 R2R3 MYB genes. Our phylogenetic analyses demonstrate, for the first time, the complex evolutionary history of the subgroup 9 R2R3 MYB genes. A duplication event is inferred before the origin of seed plants giving rise to two major gene lineages, here termed SBG9-A and SBG9-B. The evolutionary conservation of the SBG9-B gene lineage has not been previously recognized and its role in cellular differentiation is unknown, thus an entire clade of potential candidate genes for epidermal cell regulation remains to be explored. Using a heterologous transformation bioassay, we provide functional data that implicate members of the SBG9-B lineage in the specification of epidermal projections. Furthermore, we reveal numerous putative duplication events in both SBG9-A and SBG9-B lineages, resolving uncertainty about orthology and paralogy among the subgroup 9 R2R3 MYB genes. Finally, we provide a robust framework over which to interpret existing functional data and to direct ongoing comparative genetic research into the evolution of plant cellular diversity.

Gradual disintegration of the floral symmetry gene network is implicated in the evolution of a wind-pollination syndrome.

Angiosperms exhibit staggering diversity in floral form, and evolution of floral morphology is often correlated with changes in pollination syndrome. The showy, bilaterally symmetrical flowers of the model species Antirrhinum majus (Plantaginaceae) are highly specialized for bee pollination. In A. majus, Cycloidea (CYC), Dichotoma (DICH), Radialis (RAD), and Divaricata (DIV) specify the development of floral bilateral symmetry. However, it is unclear to what extent evolution of these genes has resulted in flower morphological divergence among closely related members of Plantaginaceae differing in pollination syndrome. We compared floral symmetry genes from insect-pollinated Digitalis purpurea, which has bilaterally symmetrical flowers, with those from closely related Aragoa abietina and wind-pollinated Plantago major, both of which have radially symmetrical flowers. We demonstrate that Plantago, but not Aragoa, species have lost a dorsally expressed CYC-like gene and downstream targets RAD and DIV. Furthermore, the single P. major CYC-like gene is expressed across all regions of the flower, similar to expression of its ortholog in closely related Veronica serpyllifolia. We propose that changes in the expression of duplicated CYC-like genes led to the evolution of radial flower symmetry in Aragoa/Plantago, and that further disintegration of the symmetry gene pathway resulted in the wind-pollination syndrome of Plantago. This model underscores the potential importance of gene loss in the evolution of ecologically important traits.

Evolution of petaloid sepals independent of shifts in B-class MADS box gene expression.

Attractive petals are an integral component of animal-pollinated flowers and in many flowering plant species are restricted to the second floral whorl. Interestingly, multiple times during angiosperm evolution, petaloid characteristics have expanded to adjacent floral whorls or to extra-floral organs. Here, we investigate developmental characteristics of petaloid sepals in Rhodochiton atrosanguineum, a close relative of the model species Antirrhinum majus (snapdragon). We undertook this in two ways, first using scanning electron microscopy we investigate the micromorphology of petals and sepals, followed by expression studies of genes usually responsible for the formation of petaloid structures. From our data, we conclude that R. atrosanguineum petaloid sepals lack micromorphological characteristics of petals and that petaloid sepals did not evolve through regulatory evolution of B-class MADS box genes, which have been shown to specify second whorl petal identity in a number of model flowering plant species including snapdragon. These data, in conjunction with other studies, suggests multiple convergent pathways for the evolution of showy sepals.

Patterns of shoot architecture in locally adapted populations are linked to intraspecific differences in gene regulation.

• Shoot architecture, including the number and location of branches, is a crucial aspect of plant function, morphological diversification, life history evolution and crop domestication. • Genes controlling shoot architecture are well characterized in, and largely conserved across, model flowering plant species. The role of these genes in the evolution of morphological diversity in natural populations, however, has not been explored. • We identify axillary meristem outgrowth as a primary driver of divergent branch number and life histories in two locally adapted populations of the monkeyflower, Mimulus guttatus. • Furthermore, we show that MORE AXILLARY GROWTH (MAX) gene expression strongly correlates with natural variation in branch outgrowth in this species, linking modification of the MAX-dependent pathway to the evolutionary diversification of shoot architecture.

A CYC-RAD-DIV-DRIF interaction likely pre-dates the origin of floral monosymmetry in Lamiales.

BACKGROUND: An outstanding question in evolutionary biology is how genetic interactions defining novel traits evolve. They may evolve either by de novo assembly of previously non-interacting genes or by en bloc co-option of interactions from other functions. We tested these hypotheses in the context of a novel phenotype-Lamiales flower monosymmetry-defined by a developmental program that relies on regulatory interaction among CYCLOIDEA, RADIALIS, DIVARICATA, and DRIF gene products. In Antirrhinum majus (snapdragon), representing Lamiales, we tested whether components of this program likely function beyond their previously known role in petal and stamen development. In Solanum lycopersicum (tomato), representing Solanales which diverged from Lamiales before the origin of Lamiales floral monosymmetry, we additionally tested for regulatory interactions in this program. RESULTS: We found that RADIALIS, DIVARICATA, and DRIF are expressed in snapdragon ovaries and developing fruit, similar to their homologs during tomato fruit development. In addition, we found that a tomato CYCLOIDEA ortholog positively regulates a tomato RADIALIS ortholog. CONCLUSION: Our results provide preliminary support to the hypothesis that the developmental program defining floral monosymmetry in Lamiales was co-opted en bloc from a function in carpel development. This expands our understanding of novel trait evolution facilitated by co-option of existing regulatory interactions.

SQUAMOSA-PROMOTER BINDING PROTEIN 1 initiates flowering in Antirrhinum majus through the activation of meristem identity genes.

The degree to which developmental genetic pathways are conserved across distantly related organisms is a major question in biology. In Arabidopsis thaliana (L.) Heynh., inflorescence development is initiated in response to a combination of external and internal floral inductive signals that are perceived across the whole plant, but are integrated within the shoot apical meristem. Recently, it was demonstrated that SQUAMOSA-PROMOTER BINDING PROTEIN (SBP)-box proteins regulate A. thaliana flowering time by mediating signals from the autonomous and photoperiod pathways, and by directly activating key genes involved in inflorescence and floral meristem identity, including FRUITFULL (FUL), APETALA1 (AP1) and LEAFY (LFY). In the distantly related core eudicot species Antirrhinum majus L., paralogous SBP-box proteins SBP1 and SBP2 have likewise been implicated in regulating the AP1 ortholog SQUAMOSA (SQUA). To test the hypothesis that SBP-box genes are also involved in the floral induction of A. majus, we used a reverse genetic approach to silence SBP1. SBP1-silenced lines are late to nonflowering, and show reduced apical dominance. Furthermore, expression and sequence analyses suggest that the SBP1-mediated transition to flowering occurs through the positive regulation of FUL/LFY homologs. Together, these data outline the utility of virus-induced gene silencing in A. majus, and provide new insight into the conservation of flowering time genetic pathways across core eudicots.

Differential regulation of a MYB transcription factor is correlated with transgenerational epigenetic inheritance of trichome density in Mimulus guttatus.

• Epigenetic inheritance, transgenerational transmission of traits not proximally determined by DNA sequence, has been linked to transmission of chromatin modifications and gene regulation, which are known to be sensitive to environmental factors. Mimulus guttatus increases trichome (plant hair) density in response to simulated herbivore damage. Increased density is expressed in progeny even if progeny do not experience damage. To better understand epigenetic inheritance of trichome production, we tested the hypothesis that candidate gene expression states are inherited in response to parental damage. • Using M. guttatus recombinant inbred lines, offspring of leaf-damaged and control plants were raised without damage. Relative expression of candidate trichome development genes was measured in offspring. Line and parental damage effects on trichome density were measured. Associations between gene expression, trichome density, and response to parental damage were determined. • We identified M. guttatus MYB MIXTA-like 8 as a possible negative regulator of trichome development. We found that parental leaf damage induces down-regulation of MYB MIXTA-like 8 in progeny, which is associated with epigenetically inherited increased trichome density. • Our results link epigenetic transmission of an ecologically important trait with differential gene expression states - providing insight into a mechanism underlying environmentally induced 'soft inheritance'.

Reduce, reuse, and recycle: developmental evolution of trait diversification.

A major focus of evolutionary developmental (evo-devo) studies is to determine the genetic basis of variation in organismal form and function, both of which are fundamental to biological diversification. Pioneering work on metazoan and flowering plant systems has revealed conserved sets of genes that underlie the bauplan of organisms derived from a common ancestor. However, the extent to which variation in the developmental genetic toolkit mirrors variation at the phenotypic level is an active area of research. Here we explore evidence from the angiosperm evo-devo literature supporting the frugal use of genes and genetic pathways in the evolution of developmental patterning. In particular, these examples highlight the importance of genetic pleiotropy in different developmental modules, thus reducing the number of genes required in growth and development, and the reuse of particular genes in the parallel evolution of ecologically important traits.