Mimulus Lewisii Research Articles

Genetic basis of nectar guide trichome variation between bumblebee- and self-pollinated monkeyflowers (Mimulus): role of the MIXTA-like gene GUIDELESS

Nectar guide trichomes play crucial ecological roles in bee-pollinated flowers, as they serve as footholds and guides for foraging bees to access the floral rewards. However, the genetic basis of natural variation in nectar guide trichomes among species remains poorly understood. In this study, we performed genetic analysis of nectar guide trichome variation between two closely related monkeyflower (Mimulus) species, the bumblebee-pollinated Mimulus lewisii and self-pollinated M. parishii. We demonstrate that a MIXTA-like R2R3-MYB gene, GUIDELESS, is a major contributor to the nectar guide trichome length variation between the two species. The short-haired M. parishii carries a recessive allele due to non-synonymous substitutions in a highly conserved motif among MIXTA-like MYB proteins. Furthermore, our results suggest that besides GUIDELESS, additional loci encoding repressors of trichome elongation also contribute to the transition from bumblebee-pollination to selfing. Taken together, these results suggest that during a pollination syndrome switch, changes in seemingly complex traits such as nectar guide trichomes could have a relatively simple genetic basis, involving just a few genes of large effects.

The leaf polarity factors SGS3 and YABBYs regulate style elongation through auxin signaling in Mimulus lewisii.

Style length is a major determinant of breeding strategies in flowering plants and can vary dramatically between and within species. However, little is known about the genetic and developmental control of style elongation. We characterized the role of two classes of leaf adaxial-abaxial polarity factors, SUPPRESSOR OF GENE SILENCING3 (SGS3) and the YABBY family transcription factors, in the regulation of style elongation in Mimulus lewisii. We also examined the spatiotemporal patterns of auxin response during style development. Loss of SGS3 function led to reduced style length via limiting cell division, and downregulation of YABBY genes by RNA interference resulted in shorter styles by decreasing both cell division and cell elongation. We discovered an auxin response minimum between the stigma and ovary during the early stages of pistil development that marks style differentiation. Subsequent redistribution of auxin response to this region was correlated with style elongation. Auxin response was substantially altered when both SGS3 and YABBY functions were disrupted. We suggest that auxin signaling plays a central role in style elongation and that the way in which auxin signaling controls the different cell division and elongation patterns underpinning natural style length variation is a major question for future research.

Comparative analysis of corolla tube development across three closely related Mimulus species with different pollination syndromes.

Fusion of petals to form a corolla tube is considered a key innovation contributing to the diversification of many flowering plant lineages. Corolla tube length often varies dramatically among species and is a major determinant of pollinator preference. However, our understanding of the developmental dynamics underlying corolla tube length variation is very limited. Here we examined corolla tube growth in the Mimulus lewisii species complex, an emerging model system for studying the developmental genetics and evo-devo of pollinator-associated floral traits. We compared developmental and cellular processes associated with corolla tube length variation among the bee-pollinated M. lewisii, the hummingbird-pollinated Mimulusverbenaceus, and the self-pollinated Mimulus parishii. We found that in all three species, cell size is non-uniformly distributed along the mature tube, with the longest cells just distal to the stamen insertion site. Differences in corolla tube length among the three species are not associated with processes of organogenesis or early development but are associated with variation in multiple processes occurring later in development, including the location and duration of cell division and cell elongation. The tube growth curves of the small-flowered M. parishii and large-flowered M. lewisii are essentially indistinguishable, except that M. parishii tubes stop growing earlier at a smaller size, suggesting a critical role of heterochrony in the shift from outcrossing to selfing. These results not only highlight the developmental process associated with corolla tube variation among species but also provide a baseline reference for future developmental genetic analyses of mutants or transgenic plants with altered corolla tube morphology in this emerging model system.

Convergence of glandular trichome morphology and chemistry in two montane monkeyflower (Mimulus, Phrymaceae) species

Two distantly-related North American montane monkeyflower species, Mimulus lewisii and M. tilingii, possess glandular trichomes. In this study, we characterized the morphological and histochemical features of these glandular trichomes. For each species, we used traditional light microscopy and scanning electron microscopy (SEM) to examine morphological characteristics, and categorized the natural products on the leaf surface using histochemical and thin layer chromatography (TLC) staining techniques. We identified type VI capitate glandular trichomes on leaf surfaces in both species of monkeyflowers. These trichomes exhibited stalk-cell lengths and head-cell counts that varied across adaxial and abaxial leaf surfaces. Both species contained secretory products within the cuticle of the trichome head, which releases the subcuticular metabolites when ruptured. Histochemical tests of M. lewisii and M. tilingii indicated the presence of lipids and polysaccharides on the leaf surface. TLC plate staining further supported their presence, as well as UV active compounds and amines. The common morphology and chemistry of the glandular trichomes suggests an analogous response to similar environmental conditions in these evolutionary distant montane monkeyflowers.

Cloning and characterization of a Mimulus lewisii NPR1 gene involved in regulating plant resistance to Rhizoctonia solani.

The monkey flower Mimulus lewisii is a new emerging model plant for the study in corolla tube formation, pigmentation patterns and pollinator selection, etc. However, the cultivation and management of this plant are difficult due to its susceptibility to a wide range of pathogens and the lack of rigid varieties with high levels of resistance to pathogens. In this regard, genetic engineering is a promising tool that may possibly allow us to enhance the M. lewisii disease resistance against pathogens. Here, we reported the isolation and characterization of non-expressor of pathogenesis related gene 1 (NPR1) gene from M. lewisii. The phylogenetic tree constructed based on the deduced sequence of MlNPR1 with homologs from other species revealed that MlNPR1 grouped together with other known NPR1 proteins of Scrophulariaceae family, and was nearest to Mimulus guttatus. Furthermore, expression analysis showed that MlNPR1 was upregulated after SA treatment and fungal infection. To understand the defensive role of this gene, we overexpressed MlNPR1 in M. lewisii. The transgenic lines showed slight phenotypic abnormalities, but constitutive expression of MlNPR1 activates defense signaling pathways by priming the expression of antifungal PR genes. Moreover, MlNPR1 transgenic lines showed enhanced resistance to Rhizoctonia solani there was delay in symptoms and reduced disease severity than non-transgenic plants. Altogether, the present study suggests that increasing the expression level of MlNPR1 may be a promising approach for development of monkey flower cultivars with enhanced resistance to diseases.

Less is more: Independent loss-of-function OCIMENE SYNTHASE alleles parallel pollination syndrome diversification in monkeyflowers (Mimulus).

Pollinator-mediated selection on flower phenotypes (e.g., shape, color, scent) is key to understanding the adaptive radiation of angiosperms, many of which have evolved specialized relationships with a particular guild of animal pollinators (e.g., birds, bats, moths, bees). E-β-Ocimene, a monoterpene produced by OCIMENE SYNTHASE (OS) in Mimulus lewisii, is a floral scent important in attracting the species' bumblebee pollinators. The taxa closely related to M. lewisii have evolved several different pollination syndromes, including hummingbird pollination and self pollination (autogamy). We are interested in how floral scent variation contributed to species diversification in this clade. We analyzed variation in E-β-ocimene emission within this Mimulus clade and explored its molecular basis through a combination of DNA sequencing, reverse transcriptase PCR, and enzyme functional analysis in vitro. We found that none of the taxa, other than M. lewisii, emitted E-β-ocimene from flowers. But the molecular basis underlying loss of E-β-ocimene emission is unique in each taxon, including deletion, missense, or frameshift mutations in the OS gene, and potential posttranscriptional downregulation. The molecular evidence suggests that parallel loss-of-function in OS is the best explanation for the observed pattern of E-β-ocimene emission, likely as the result of natural selection.

Testing the utility of fluorescent proteins in Mimulus lewisii by an Agrobacterium-mediated transient assay.

The Agrobacterium -mediated transient expression assay by leaf infiltration in Mimulus lewisii is robust. Fluorescent proteins EGFP, EYFP and DsRed give bright fluorescence signals in the infiltrated tissue. Mimulus lewisii is an emerging developmental genetic model system. Recently developed genomic and genetic resources and a stable transformation protocol have greatly facilitated the identification and functional characterization of genes controlling the development of ecologically important floral traits using this species. To further expedite gene and protein function analyses in M. lewisii, we adopted and simplified the Agrobacterium-mediated transient gene expression method routinely used in tobacco plants. With the validated transient assay, we examined the performance of fluorescent proteins EGFP, EYFP and DsRed in M. lewisii. All three proteins gave bright fluorescence signals when transiently expressed in agroinfiltrated leaves. Furthermore, we demonstrated the utility of fluorescent proteins in M. lewisii by showing the nuclear localization of Reduced Carotenoid Pigmentation 1 (RCP1), a recently discovered R2R3-MYB transcription factor that regulates carotenoid pigmentation during flower development. Both the transient assay and the fluorescent proteins are valuable additions to the M. lewisii toolbox, making this emerging genetic and developmental model system even more powerful.

Carotenoid composition of the flowers of Mimulus lewisii and related species: Implications regarding the prevalence and origin of two unique, allenic pigments

The genus Mimulus has been used as a model system in a wide range of ecological and evolutionary studies and contains many species with carotenoid pigmented flowers. However, the detailed carotenoid composition of these flowers has never been reported. In this paper the floral carotenoid composition of 11 Mimulus species are characterized using high-performance liquid chromatography, mass spectrometry and chemical methods with a particular focus on the genetic model species, Mimulus lewisii. M. lewisii flowers have five major carotenoids: antheraxanthin, violaxanthin, neoxanthin, and the unique allenic carotenoids, deepoxyneoxanthin and mimulaxanthin. This carotenoid profile is consistent with the expression levels of putative carotenoid biosynthetic genes in the M. lewisii flower. The other 10 species possess the same five carotenoids or a subset of these. Comparison of the carotenoid profiles among species in a phylogenetic context provides new insights into the biosynthesis and evolution of deepoxyneoxanthin and mimulaxanthin. This work also lays the foundation for future studies regarding transcriptional control of the carotenoid biosynthesis pathway in Mimulus flowers.

Floral volatile alleles can contribute to pollinator-mediated reproductive isolation in monkeyflowers (Mimulus).

Pollinator-mediated reproductive isolation is a major factor in driving the diversification of flowering plants. Studies of floral traits involved in reproductive isolation have focused nearly exclusively on visual signals, such as flower color. The role of less obvious signals, such as floral scent, has been studied only recently. In particular, the genetics of floral volatiles involved in mediating differential pollinator visitation remains unknown. The bumblebee-pollinated Mimulus lewisii and hummingbird-pollinated Mimulus cardinalis are a model system for studying reproductive isolation via pollinator preference. We have shown that these two species differ in three floral terpenoid volatiles - d-limonene, β-myrcene, and E-β-ocimene - that are attractive to bumblebee pollinators. By genetic mapping and in vitro analysis of enzyme activity we demonstrate that these interspecific differences are consistent with allelic variation at two loci, LIMONENE-MYRCENE SYNTHASE (LMS) and OCIMENE SYNTHASE (OS). Mimulus lewisii LMS (MlLMS) and OS (MlOS) are expressed most strongly in floral tissue in the last stages of floral development. Mimulus cardinalis LMS (McLMS) is weakly expressed and has a nonsense mutation in exon 3. Mimulus cardinalis OS (McOS) is expressed similarly to MlOS, but the encoded McOS enzyme produces no E-β-ocimene. Recapitulating the M.cardinalis phenotype by reducing the expression of MlLMS by RNA interference in transgenic M.lewisii produces no behavioral difference in pollinating bumblebees; however, reducing MlOS expression produces a 6% decrease in visitation. Allelic variation at the OCIMENE SYNTHASE locus is likely to contribute to differential pollinator visitation, and thus promote reproductive isolation between M.lewisii and M.cardinalis. OCIMENE SYNTHASE joins a growing list of 'speciation genes' ('barrier genes') in flowering plants.

The genetic architecture of traits associated with the evolution of self-pollination in Mimulus.

Quantitative trait locus (QTL) mapping is a first step toward understanding the genetic basis of adaptive evolution and may also reveal reproductive incompatibilities unique to hybrids. In plants, the shift from outcrossing to self-pollination is common, providing the opportunity for comparisons of QTL architecture among parallel evolutionary transitions. We used QTL mapping in hybrids between the bee-pollinated monkeyflower Mimulus lewisii and the closely related selfer Mimulus parishii to determine the genetic basis of divergence in floral traits and flowering time associated with mating-system evolution, and to characterize hybrid anther sterility. We found a moderately polygenic and highly directional basis for floral size evolution, suggesting adaptation from standing variation or in pursuit of a moving optimum, whereas only a few major loci accounted for substantial flowering-time divergence. Cytonuclear incompatibilities caused hybrid anther sterility, confounding estimation of reproductive organ QTLs. The genetic architecture of floral traits associated with selfing in M. parishii was primarily polygenic, as in other QTL studies of this transition, but in contrast to the previously characterized oligogenic basis of a pollinator shift in close relatives. Hybrid anther sterility appeared parallel at the molecular level to previously characterized incompatibilities, but also raised new questions about cytonuclear co-evolution in plants.

Chromosomal rearrangements directly cause underdominant F1 pollen sterility in Mimulus lewisii-Mimulus cardinalis hybrids.

Chromosomal rearrangements can contribute to the evolution of postzygotic reproductive isolation directly, by disrupting meiosis in F1 hybrids, or indirectly, by suppressing recombination among genic incompatibilities. Because direct effects of rearrangements on fertility imply fitness costs during their spread, understanding the mechanism of F1 hybrid sterility is integral to reconstructing the role(s) of rearrangements in speciation. In hybrids between monkeyflowers Mimulus cardinalis and Mimulus lewisii, rearrangements contain all quantitative trait loci (QTLs) for both premating barriers and pollen sterility, suggesting that they may have facilitated speciation in this model system. We used artificial chromosome doubling and comparative mapping to test whether heterozygous rearrangements directly cause underdominant male sterility in M. lewisii-M. cardinalis hybrids. Consistent with a direct chromosomal basis for hybrid sterility, synthetic tetraploid F1 s showed highly restored fertility (83.4% pollen fertility) relative to diploids F1 s (36.0%). Additional mapping with Mimulus parishii-M. cardinalis and M. parishii-M. lewisii hybrids demonstrated that underdominant male sterility is caused by one M. lewisii specific and one M. cardinalis specific reciprocal translocation, but that inversions had no direct effects on fertility. We discuss the importance of translocations as causes of reproductive isolation, and consider models for how underdominant rearrangements spread and fix despite intrinsic fitness costs.

Transcriptional control of floral anthocyanin pigmentation in monkeyflowers (Mimulus).

A molecular description of the control of floral pigmentation in a multi-species group displaying various flower color patterns is of great interest for understanding the molecular bases of phenotypic diversification and pollinator-mediated speciation. Through transcriptome profiling, mutant analyses and transgenic experiments, we aim to establish a 'baseline' floral anthocyanin regulation model in Mimulus lewisii and to examine the different ways of tinkering with this model in generating the diversity of floral anthocyanin patterns in other Mimulus species. We find one WD40 and one bHLH gene controlling anthocyanin pigmentation in the entire corolla of M.lewisii and two R2R3-MYB genes, PELAN and NEGAN, controlling anthocyanin production in the petal lobe and nectar guide, respectively. The autoregulation of NEGAN might be a critical property to generate anthocyanin spots. Independent losses of PELAN expression (via different mechanisms) explain two natural yellow-flowered populations of M.cardinalis (typically red-flowered). The NEGAN ortholog is the only anthocyanin-activating MYB expressed in the M.guttatus flowers. The mutant lines and transgenic tools available for M.lewisii will enable gene-by-gene replacement experiments to dissect the genetic and developmental bases of more complex floral color patterns, and to test hypotheses on phenotypic evolution in general.

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.

CHROMOSOMAL REARRANGEMENTS AND THE GENETICS OF REPRODUCTIVE BARRIERS INMIMULUS(MONKEY FLOWERS)

Chromosomal rearrangements may directly cause hybrid sterility and can facilitate speciation by preserving local adaptation in the face of gene flow. We used comparative linkage mapping with shared gene-based markers to identify potential chromosomal rearrangements between the sister monkeyflowers Mimulus lewisii and Mimulus cardinalis, which are textbook examples of ecological speciation. We then remapped quantitative trait loci (QTLs) for floral traits and flowering time (premating isolation) and hybrid sterility (postzygotic isolation). We identified three major regions of recombination suppression in the M. lewisii × M. cardinalis hybrid map compared to a relatively collinear Mimulus parishii × M. lewisii map, consistent with a reciprocal translocation and two inversions specific to M. cardinalis. These inferences were supported by targeted intraspecific mapping, which also implied a M. lewisii-specific reciprocal translocation causing chromosomal pseudo-linkage in both hybrid mapping populations. Floral QTLs mapped in this study, along with previously mapped adaptive QTLs, were clustered in putatively rearranged regions. All QTLs for male sterility, including two underdominant loci, mapped to regions of recombination suppression. We argue that chromosomal rearrangements may have played an important role in generating and consolidating barriers to gene flow as natural selection drove the dramatic ecological and morphological divergence of these species.

Genetic Dissection of a Major Anthocyanin QTL Contributing to Pollinator-Mediated Reproductive Isolation Between Sister Species ofMimulus

Prezygotic barriers play a major role in the evolution of reproductive isolation, which is a prerequisite for speciation. However, despite considerable progress in identifying genes and mutations responsible for postzygotic isolation, little is known about the genetic and molecular basis underlying prezygotic barriers. The bumblebee-pollinated Mimulus lewisii and the hummingbird-pollinated M. cardinalis represent a classic example of pollinator-mediated prezygotic isolation between two sister species in sympatry. Flower color differences resulting from both carotenoid and anthocyanin pigments contribute to pollinator discrimination between the two species in nature. Through fine-scale genetic mapping, site-directed mutagenesis, and transgenic experiments, we demonstrate that a single-repeat R3 MYB repressor, ROSE INTENSITY1 (ROI1), is the causal gene underlying a major quantitative trait locus (QTL) with the largest effect on anthocyanin concentration and that cis-regulatory change rather than coding DNA mutations cause the allelic difference between M. lewisii and M. cardinalis. Together with the genomic resources and stable transgenic tools developed here, these results suggest that Mimulus is an excellent platform for studying the genetics of pollinator-mediated reproductive isolation and the molecular basis of morphological evolution at the most fundamental level-gene by gene, mutation by mutation.

Three floral volatiles contribute to differential pollinator attraction in monkeyflowers (Mimulus)

Flowering plants employ a wide variety of signals, including scent, to attract the attention of pollinators. In this study we investigated the role of floral scent in mediating differential attraction between two species of monkeyflowers (Mimulus) reproductively isolated by pollinator preference. The emission rate and chemical identity of floral volatiles differ between the bumblebee-pollinated Mimulus lewisii and the hummingbird-pollinated M. cardinalis. Mimulus lewisii flowers produce an array of volatiles dominated by d-limonene, β-myrcene and E-β-ocimene. Of these three monoterpenes, M. cardinalis flowers produce only d-limonene, released at just 0.9% the rate of M. lewisii flowers. Using the Bombus vosnesenskii bumblebee, an important pollinator of M. lewisii, we conducted simultaneous gas chromatography with extracellular recordings in the bumblebee antennal lobe. Results from these experiments revealed that these three monoterpenes evoke significant neural responses, and that a synthetic mixture of the three volatiles evokes the same responses as the natural scent. Furthermore, the neural population shows enhanced responses to the M. lewisii scent over the scent of M. cardinalis. This neural response is reflected in behavior; in two-choice assays, bumblebees investigate artificial flowers scented with M. lewisii more frequently than ones scented with M. cardinalis, and in synthetic mixtures the three monoterpenes are necessary and sufficient to recapitulate responses to the natural scent of M. lewisii. In this system, floral scent alone is sufficient to elicit differential visitation by bumblebees, implying a strong role of scent in the maintenance of reproductive isolation between M. lewisii and M. cardinalis.

Gone with the wind: understanding evolutionary transitions between wind and animal pollination in the angiosperms

Gone with the wind: understanding evolutionary transitions between wind and animal pollination in the angiosperms

Induced mutations affecting pollinator choice in Mimulus lewisii (Phrymaceae)

The flowering plants are one of the most phenotypically varied and wide-ranging groups of organisms on earth, and yet, we have limited understanding of the contribution of animal pollinators to the diversification of floral form. To explore the interaction between variation in floral form and pollinator behavior, we observed the foraging behavior of bumblebees (Bombus impatiens) when presented with both wild-type Mimulus lewisii plants and each of three chemically induced single-locus mutants with altered floral phenotypes, including loss of the three lower petals, loss of nectar guides, and a change in petal color patterning. We found that each of the mutants attracted successful pollinator visits at just 29–80% of the rate relative to wild-type flowers, suggesting that effective recruitment of bumblebee pollinators requires the landing platform provided by the lower petals, and visual cues provided by the nectar guides and petal color pattern. Since single-locus recessive mutations are capable of ablating the lower petals, nectar guides, and color pattern, such changes in floral form provide insight into the driving forces behind plant adaptation.

Identification of microRNAs in ecological model plant <i>Mimulus</i>

MicroRNAs (miRNAs) are small, non-coding and regulatory RNAs about 20 to 24 nucleotides long. Their conserved nature among the various organisms makes them a good source of new miRNAs discovery by comparative genomics approach. This study resulted in 34 miRNAs belonging to 21 families in ecological model plant Mimulus. All the miRNA families (mir 156, 157, 159, 160, 164, 172, 319, 393, 395, 397, 399, 400, 403, 417, 419, 472, 782, 854, 858, 867 and 2112) are found for the first time in Mimulus. The MIR-399 is found as precursor miRNA cluster with 4 mature sequences. All 34 miRNA precursors form stable minimum free energy stem loop structure as their orthologues form and the mature miRNAs reside in the stem portion of the stem loop structure. Twenty eight are from Mimulus guttatus and six miRNAs belong to Mimulus lewisii. Their targets consist of dihy-droflavonol-4-reductase, cycloidea-like protein, DNA-directed RNA polymerase II, maturase (matR) and transcription factors like; squamosa-promoter binding, MYB, palmate-like pentafoliata 1.

The niche, limits to species' distributions, and spatiotemporal variation in demography across the elevation ranges of two monkeyflowers.

Understanding the processes that create and maintain species' geographic range limits has implications for many questions in ecology, evolution, and conservation biology. Many expectations for the ecological and evolutionary dynamics of populations at the range margin rest on the concordance of geographic limits and the limits of a species' ecological niche. If range limits are coincident with niche limits, then marginal populations should have lower and/or more variable vital rates and population growth rates than central populations. Using data from 8 annual censuses of marked individuals, I investigated the demography of Mimulus cardinalis and Mimulus lewisii across the species' elevation ranges. Central and marginal populations exhibited striking demographic differences, but only for one species were differences in expected directions. Marginal populations from the M. lewisii lower elevation range limit had lower and more variable survival than central populations and appeared to be demographic sinks. In contrast, marginal populations from the M. cardinalis upper elevation limit had higher fecundity and higher population growth rates than central populations. Although the species differed with respect to central-marginal patterns, they were concordant with respect to elevation; that is, both species had higher fitness in higher reaches of their examined ranges. Potential explanations for these patterns include source-sink dynamics, with asymmetrical gene flow mediated by river currents, and climate change, with recent warming shifting the species' climatic envelopes to higher elevations. Hence, assessment of spatiotemporal variation in both demography and dispersal is necessary to fully understand the relationship between the niche and species' distributions.