The timing of visits by large and small bees differentially affects pollination success in Mimulus ringens.

PREMISE: Cross-fertilization in most flowering plants is facilitated by mobile animals that transport pollen while foraging for floral rewards. The contributions of different visitors can vary widely, depending on the amount of pollen transferred during a single visit and on the frequency and timing of the visits of each pollinator taxon. METHODS: We used three approaches to measure the pollination value of bees that visit Mimulus ringens: pollinator interviews, field population observations, and caging studies. RESULTS: The single-visit effectiveness of small bees (primarily Halictidae) was only half that of larger bees (primarily Bombus) for pollen delivery and removal. In five field populations, we found substantial temporal and spatial variation in visitation and pollination. In most sites big bees were active before 08:00 hours, and by 10:00-11:00 hours, stigmas were usually fully pollinated and closed, and little pollen remained in anthers. Small bees seldom visited before 10:00 hours. Excluding big bees from plants confirmed that pollination is reduced and delayed in this ecological context. CONCLUSIONS: Big bees are the primary pollinators of M. ringens, accounting for at least 75% of seed production. Not only are they more effective per visit, in most situations they also visit before small bees become active. Although small bees are not usually important pollinators of M. ringens, they have the potential to partially replace them as a "fail-safe" pollinator in contexts where big bees are not abundant. In a world where pollinator abundance is declining, such backup pollinators may be important for maintaining plant reproduction.

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).

Comparative plastomes of species from Phrymaceae and Mazaceae: insights into adaptive evolution, codon usage bias, and phylogenetic relationships.

The phylogeny of the species from Phrymaceae and Mazaceae has undergone many adjustments and changes in recent years. Moreover, there is little plastome information on the Phrymaceae. In this study, we compared the plastomes of six species from the Phrymaceae and 10 species from the Mazaceae. The gene order, contents, and orientation of the 16 plastomes were found to be highly similar. A total of 13 highly variable regions were identified among the 16 species. An accelerated rate of substitution was found in the protein-coding genes, particularly cemA and matK. The combination of effective number of codons, parity rule 2, and neutrality plots revealed that the codon usage bias is affected by mutation and selection. The phylogenetic analysis strongly supported {Mazaceae [(Phrymaceae + Wightiaceae) + (Paulowniaceae + Orobanchaceae)]} relationships in the Lamiales. Our findings can provide useful information to analyze the phylogeny and molecular evolution within the Phrymaceae and Mazaceae.

Selection on early survival does not explain germination rate clines in Mimulus cardinalis.

PREMISE: Many traits covary with environmental gradients to form phenotypic clines. While local adaptation to the environment can generate phenotypic clines, other nonadaptive processes may also. If local adaptation causes phenotypic clines, then the direction of genotypic selection on traits should shift from one end of the cline to the other. Traditionally, genotypic selection on non-Gaussian traits like germination rate have been hampered because it is challenging to measure their genetic variance. METHODS: Here we used quantitative genetics and reciprocal transplants to test whether a previously discovered cline in germination rate showed additional signatures of adaptation in the scarlet monkeyflower (Mimulus cardinalis). We measured genotypic and population level covariation between germination rate and early survival, a component of fitness. We developed a novel discrete log-normal model to estimate genetic variance in germination rate. RESULTS: Contrary to our adaptive hypothesis, we found no evidence that genetic variation in germination rate contributed to variation in early survival. Across populations, southern populations in both gardens germinated earlier and survived more. CONCLUSIONS: Southern populations have higher early survival but it is not caused by faster germination. This pattern is consistent with nonadaptive forces driving the phenotypic cline in germination rate, but future work will need to assess whether there is selection at other life stages. This statistical framework should help expand quantitative genetic analyses for other waiting-time traits.

Quantitative trait locus mapping reveals an independent genetic basis for joint divergence in leaf function, life-history, and floral traits between scarlet monkeyflower (Mimulus cardinalis) populations.

PREMISE: Across taxa, vegetative and floral traits that vary along a fast-slow life-history axis are often correlated with leaf functional traits arrayed along the leaf economics spectrum, suggesting a constrained set of adaptive trait combinations. Such broad-scale convergence may arise from genetic constraints imposed by pleiotropy (or tight linkage) within species, or from natural selection alone. Understanding the genetic basis of trait syndromes and their components is key to distinguishing these alternatives and predicting evolution in novel environments. METHODS: We used a line-cross approach and quantitative trait locus (QTL) mapping to characterize the genetic basis of twenty leaf functional/physiological, life history, and floral traits in hybrids between annualized and perennial populations of scarlet monkeyflower (Mimulus cardinalis). RESULTS: We mapped both single and multi-trait QTLs for life history, leaf function and reproductive traits, but found no evidence of genetic co-ordination across categories. A major QTL for three leaf functional traits (thickness, photosynthetic rate, and stomatal resistance) suggests that a simple shift in leaf anatomy may be key to adaptation to seasonally dry habitats. CONCLUSIONS: Our results suggest that the co-ordination of resource-acquisitive leaf physiological traits with a fast life-history and more selfing mating system results from environmental selection rather than functional or genetic constraint. Independent assortment of distinct trait modules, as well as a simple genetic basis to leaf physiological traits associated with drought escape, may facilitate adaptation to changing climates.

Population responses to a historic drought across the range of the common monkeyflower (Mimulus guttatus).

PREMISE: Due to climate change, more frequent and intense periodic droughts are predicted to increasingly pose major challenges to the persistence of plant populations. When a severe drought occurs over a broad geographical region, independent responses by individual populations provide replicated natural experiments for examining the evolution of drought resistance and the potential for evolutionary rescue. METHODS: We used a resurrection approach to examine trait evolution in populations of the common monkeyflower, Mimulus guttatus, exposed to a record drought in California from 2011 to 2017. Specifically, we compared variation in traits related to drought escape and avoidance from seeds collected from 37 populations pre- and post-drought in a common garden. In a parallel experiment, we evaluated fitness in two populations, one which thrived and one which was nearly extirpated during the drought, under well-watered and dry-down conditions. RESULTS: We observed substantial variation among populations in trait evolution. In the subset of populations where phenotypes changed significantly, divergence proceeded along trait correlations with some populations flowering rapidly with less vegetative tissue accumulation and others delaying flowering with greater vegetative tissue accumulation. The degree of trait evolution was only weakly correlated with drought intensity but strongly correlated with initial levels of standing variation. Fitness was higher in the post-drought than pre-drought accessions in both treatments for the thriving population, but lower in both treatments for the nearly extirpated population. CONCLUSIONS: Together, our results indicate that evolutionary responses to drought are context dependent and reflect the standing genetic variation and genetic correlations present within populations.

Contrasting environmental factors drive local adaptation at opposite ends of an environmental gradient in the yellow monkeyflower (Mimulus guttatus).

PREMISE: Identifying the environmental factors responsible for natural selection across different habitats is crucial for understanding the process of local adaptation in plants. Despite its importance, few studies have successfully isolated the environmental factors driving local adaptation in nature. In this study, we evaluated the agents of selection responsible for local adaptation of the monkeyflower Mimulus guttatus to California's coastal and inland habitats. METHODS: We implemented a manipulative reciprocal transplant experiment at coastal and inland sites, where we excluded aboveground stressors in an effort to elucidate their role in the evolution of local adaptation. RESULTS: Excluding aboveground stressors, most likely a combination of salt spray and herbivory, completely rescued inland annual plant fitness when transplanted to coastal habitat. The exclosures in inland habitat provided a benefit to the performance of coastal perennial plants. However, the exclosures are unlikely to provide much fitness benefit to the coastal plants at the inland site because of their general inability to flower in time to escape from the summer drought. CONCLUSIONS: Our study demonstrates that a distinct set of selective agents (aboveground vs. belowground) are responsible for local adaptation at opposite ends of an environmental gradient.

Genomewide variation provides insight into evolutionary relationships in a monkeyflower species complex (Mimulus sect. Diplacus).

PREMISE OF THE STUDY: Evolutionary radiations provide excellent opportunities to study the origins of biodiversity, but rapid divergence and ongoing gene flow make inferring evolutionary relationships among taxa difficult. Consequently, combining morphological and genomic analyses will be necessary to clarify the evolutionary history of radiations. We used an integrative approach to shed light on relationships within a diverse radiation of monkeyflowers (Mimulus section Diplacus) with a controversial taxonomic history. METHODS: We used genomewide single nucleotide polymorphism data and a combination of phylogenetic and population genomic analyses to infer the evolutionary relationships within the group. Tests for hybridization were performed to reveal sources of shared variation, and multivariate analyses of floral trait data were conducted to examine the correspondence between phenotypic and phylogenetic data. KEY RESULTS: We identified four primary clades with evidence for some shared variation among them. We also detected evidence for recent gene flow between closely related subclades and populations. Strong discordance between floral trait and molecular data provides evidence for divergent and convergent phenotypic evolution. CONCLUSIONS: Mimulus section Diplacus has all the hallmarks of a rapid radiation, including diverse taxa that are at different stages of divergence, extensive shared variation among taxa, and complex patterns of phenotypic evolution. Our findings will direct future evolutionary research and have important taxonomic implications that highlight the need for a new revision of section Diplacus.

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

PREMISE OF THE STUDY: 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-ß-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. METHODS: We analyzed variation in E-ß-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. KEY RESULTS: We found that none of the taxa, other than M. lewisii, emitted E-ß-ocimene from flowers. But the molecular basis underlying loss of E-ß-ocimene emission is unique in each taxon, including deletion, missense, or frameshift mutations in the OS gene, and potential posttranscriptional downregulation. CONCLUSIONS: The molecular evidence suggests that parallel loss-of-function in OS is the best explanation for the observed pattern of E-ß-ocimene emission, likely as the result of natural selection.

Losing one's touch: Evolution of the touch-sensitive stigma in the Mimulus guttatus species complex.

PREMISE OF THE STUDY: The stigmas of several species are touch sensitive and respond to pressure by closing. Previous research suggests that stigma closure could prevent self pollination within a flower during a pollinator's visit or enhance male function by increasing pollen export. Both factors could be favored in outcrossers, and neither would be beneficial in selfers. METHODS: We investigated variation in stigma-closing and the duration of closure in annual and perennial populations of the variable species Mimulus guttatus and whether four closely related selfing species (M. cupriphilus, M. laciniatus, M. nasutus, and M. pardalis) have lost their touch sensitivity. We grew plants in a controlled environment and performed experiments with and without the addition of pollen to the stigma. KEY RESULTS: In M. guttatus, the speed of stigma-closing was rapid and unaffected by the deposition of pollen. Populations varied significantly in closing speed, which may reflect their geographic location. For annual populations only, anther-stigma separation significantly affected closing speed. Also, stigmas that closed quickly stayed closed longer, and stigmas that received pollen remained closed longer. Finally, in the selfing species, stigma-closing was more variable; some populations have entirely lost the ability to respond to touch. CONCLUSIONS: We discuss our results in the context of traits that promote outcrossing and traits that are under selection during the evolution of selfing. This is the first characterization of variation in touch responses across multiple populations within a species and the first to demonstrate the loss of touch sensitivity in selfing lineages.

A first test of elemental allelopathy via heterospecific pollen receipt.

PREMISE OF THE STUDY: Coflowering plants often share pollinators and may receive mixed species pollen loads. Although detrimental effects of heterospecific pollen receipt have been documented, trait-based modifiers of interactions on the stigma remain largely unknown. Chemicals that mediate interactions between sporophytes could also influence pollen-pollen or pollen-style interactions. We test for the first time whether nickel (Ni) accumulation in pollen can lead to "elemental allelopathy" and intensify the fitness consequences of heterospecific pollen receipt. METHODS: We grew Ni-hyperaccumulator Streptanthus polygaloides in soils augmented with three concentrations of Ni, measured pollen Ni concentration, and hand-pollinated non-Ni hyperaccumulator Mimulus guttatus. We assayed pollen germination, tube growth and seeds of M. guttatus after pure and mixed species pollinations. KEY RESULTS: Streptanthus polygaloides pollen accumulated Ni in proportion to soil availability and at levels significantly greater than M. guttatus pollen. Although receipt of S. polygaloides pollen increased M. guttatus pollen germination, it decreased the proportion of pollen tubes reaching the ovary and seed number. Increased Ni in pollen, however, did not significantly intensify the effect of S. polygaloides pollen receipt on M. guttatus seed production. CONCLUSIONS: Different levels of Ni in the pollen of S. polygaloides achieved in the greenhouse did not significantly reduce the fitness of M. guttatus. Stigma tolerance to Ni may also have contributed to the lack of response to increased Ni in heterospecific pollen. This study paves the way for additional tests in other metal hyperaccumulators and recipients, and to identify mechanisms of interactions on the stigma.

Absence of postmating barriers between a selfing vs. outcrossing Chilean Mimulus species pair.

PREMISE OF THE STUDY: Reproductive isolation between sympatric species pairs may be maintained by both pre- and postmating barriers. Here we evaluate potential barriers to mating between the outcrossing Mimulus luteus and its more highly selfing sympatric congener, M. cupreus, two members of the South American luteus complex of Mimulus. METHODS: Seed set was compared following autonomous self-pollination, manual pollination, conspecific outcrossing, and sympatric and allopatric hybridization, for laboratory-maintained inbred lines and wild-collected accessions. Survival and reproductive fitness of hybrids relative to parental species were examined across environments that differed with respect to temperature and soil nutrients, two factors that vary across the ranges of M. luteus and M. cupreus. KEY RESULTS: Mimulus luteus was minimally capable of autonomous self-fertilization, consistent with reliance on an animal pollinator, whereas M. cupreus was a successful selfer across all tested accessions. Postmating barriers to hybridization are negligible, in both low- and high-stress environments, across multiple sympatric and allopatric populations. CONCLUSION: As in the North American M. guttatus-M. nasutus species pair, postmating barriers contribute little to isolation between M. luteus and M. cupreus. This result reinforces the importance of premating barriers, specifically species differences in reliance on, and accessibility to, animal pollinators. A unique aspect of the M. luteus-M. cupreus pair is the recent gain of red floral anthocyanin pigmentation in M. cupreus. On the basis of species differences in vegetative anthocyanin production, a facultative stress-protective response, we propose a potential stress-protective role for the constitutive floral anthocyanins of M. cupreus.

An ultraviolet floral polymorphism associated with life history drives pollinator discrimination in Mimulus guttatus.

UNLABELLED: • PREMISE OF THE STUDY: Ultraviolet (UV) floral patterns are common in angiosperms and mediate pollinator attraction, efficiency, and constancy. UV patterns may vary within species, yet are cryptic to human observers. Thus, few studies have explicitly described the distribution or ecological significance of intraspecific variation in UV floral patterning. Here, we describe the geographic distribution and pattern of inheritance of a UV polymorphism in the model plant species Mimulus guttatus (Phrymaceae). We then test whether naturally occurring UV phenotypes influence pollinator interactions within M. guttatus.• METHODS: We document UV patterns in 18 annual and 19 perennial populations and test whether UV pattern is associated with life history. To examine the pattern of inheritance, we conducted crosses within and between UV phenotypes. Finally, we tested whether bee pollinators discriminate among naturally occurring UV phenotypes in two settings: wild bee communities and captive Bombus impatiens.• KEY RESULTS: Within M. guttatus, perennial populations exhibit a small bulls-eye pattern, whereas a bilaterally symmetric runway pattern occurs mainly in annual populations. Inheritance of UV patterning is consistent with a single-locus Mendelian model in which the runway phenotype is dominant. Bee pollinators discriminate against unfamiliar UV patterns in both natural and controlled settings.• CONCLUSIONS: We describe a widespread UV polymorphism associated with life history divergence within Mimulus guttatus. UV pattern influences pollinator visitation and should be considered when estimating reproductive barriers between life history ecotypes. This work develops a new system to investigate the ecology and evolution of UV floral patterning in a species with extensive genomic resources.

Development of SSR markers for a Tibetan medicinal plant, Lancea tibetica (Phrymaceae), based on RAD sequencing.

PREMISE OF THE STUDY: Lancea tibetica (Phrymaceae), a Tibetan medicinal plant, is endemic to the Qinghai-Tibet Plateau. The over-exploitation of wild L. tibetica has led to the destruction of many populations. To enhance protection and management, biological research, especially population genetic studies, should be carried out on L. tibetica. Simple sequence repeat (SSR) markers of L. tibetica were developed to analyze population diversity. METHODS AND RESULTS: Four thousand four hundred and forty-one SSR loci were identified for L. tibetica based on restriction-site associated DNA (RAD) sequencing on the Illumina HiSeq platform. One hundred SSR loci were arbitrarily selected for primer design, and 38 of them were successfully amplified. These markers were tested on 56 individuals from three populations of L. tibetica, and 10 markers displayed polymorphisms. The total number of alleles per locus ranged from three to eight, and observed and expected heterozygosities ranged from 0.200 to 1.000 and 0.683 to 0.879, respectively. We tested for cross-amplification of these 10 markers in the related species L. hirsuta and found that nine could be successfully amplified. CONCLUSIONS: The SSR markers characterized here are the first to be developed and tested in L. tibetica. They will be useful for future population genetic studies on L. tibetica and closely related species.