Hybrid Mimulus flowers attract a new pollinator.

Hybridization is common in flowering plants and is believed to be an important force driving adaptation and speciation. The flowers of hybrids often exhibit new trait combinations, which, theoretically, could attract new species of pollinators. In this study, we found that the hybrids between a hummingbird-pollinated species Mimulus cardinalis and a self-pollinated species Mimulus parishii attract bumblebees (Bombus impatiens), a pollinator not attracted to either of the progenitor species. This novel attraction is explained by new combinations of floral traits in hybrids, including, most importantly, petal color, in addition to nectar concentration and corolla size. To understand how petal color variation is perceived by bumblebees, we performed reflectance spectroscopy and multispectral imaging to model the flower appearance in bee vision. This analysis showed that color variation would impact the ease of detection. We also found that YUP, the genetic locus responsible for a large portion of floral color variation and previously shown to be important in bee interactions with other Mimulus species, also played an important role in this novel attraction. These results together suggest that the attraction of new pollinators to hybrid plants could be an underexplored avenue for pollinator shift and speciation.

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.

Postzygotic barriers persist despite ongoing introgression in hybridizing Mimulus species.

The evolution of postzygotic isolation is thought to be a key step in maintaining species boundaries upon secondary contact, yet the dynamics and persistence of hybrid incompatibilities in naturally hybridizing species are not well understood. Here, we explore these issues using genetic mapping in three independent populations of recombinant inbred lines between naturally hybridizing monkeyflowers, Mimulus guttatus and Mimulus nasutus, from the sympatric Catherine Creek population. We discover that the three M. guttatus founders differ dramatically in admixture history, with nearly a quarter of one founder's genome introgressed from M. nasutus. Comparative genetic mapping in the three RIL populations reveals three new putative inversions, each one segregating among the M. guttatus founders, two due to admixture. We find strong, genome-wide transmission ratio distortion in all RILs, but patterns are highly variable among the three populations. At least some of this distortion appears to be explained by epistatic selection favouring parental genotypes, but tests of inter-chromosomal linkage disequilibrium also reveal multiple candidate Dobzhansky-Muller incompatibilities. We also map several genetic loci for hybrid pollen viability, including two interacting pairs that coincide with peaks of distortion. Remarkably, even with this limited sample of three M. guttatus lines, we discover abundant segregating variation for hybrid incompatibilities with M. nasutus, suggesting this population harbours diverse contributors to postzygotic isolation. Moreover, even with substantial admixture, hybrid incompatibilities between Mimulus species persist, suggesting postzygotic isolation might be a potent force in maintaining species barriers in this system.

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).

The genetic basis of traits associated with the evolution of serpentine endemism in monkeyflowers.

The floras on chemically and physically challenging soils, such as gypsum, shale, and serpentine, are characterized by narrowly endemic species. The evolution of edaphic endemics may be facilitated or constrained by genetic correlations among traits contributing to adaptation and reproductive isolation across soil boundaries. The yellow monkeyflowers in the Mimulus guttatus species complex are an ideal system in which to examine these evolutionary patterns. To determine the genetic basis of adaptive and prezygotic isolating traits, we performed genetic mapping experiments with F2 hybrids derived from a cross between a serpentine endemic, M. nudatus, and its close relative M. guttatus. Few large effect and many small effect QTL contribute to interspecific divergence in life history, floral, and leaf traits, and a history of directional selection contributed to trait divergence. Loci contributing to adaptive traits and prezygotic reproductive isolation overlap, and their allelic effects are largely in the direction of species divergence. These loci contain promising candidate genes regulating flowering time and plant organ size. Together, our results suggest that genetic correlations among traits can facilitate the evolution of adaptation and speciation and may be a common feature of the genetic architecture of divergence between edaphic endemics and their widespread relatives.

A Multigenerational Turing Model Reproduces Transgressive Petal Spot Phenotypes in Hybrid Mimulus.

The origin of phenotypic novelty is a perennial question of genetics and evolution. To date, few studies of biological pattern formation specifically address multi-generational aspects of inheritance and phenotypic novelty. For quantitative traits influenced by many segregating alleles, offspring phenotypes are often intermediate to parental values. In other cases, offspring phenotypes can be transgressive to parental values. For example, in the model organism Mimulus (monkeyflower), the offspring of parents with solid-colored petals exhibit novel spotted petal phenotypes. These patterns are controlled by an activator-inhibitor gene regulatory network with a small number of loci. Here we develop and analyze a model of hybridization and pattern formation that accounts for the inheritance of a diploid gene regulatory network composed of either homozygous or heterozygous alleles. We find that the resulting model of multi-generational Turing-type pattern formation can reproduce transgressive petal phenotypes similar to those observed in Mimulus. The model gives insight into how non-patterned parent phenotypes can yield phenotypically transgressive, patterned offspring, aiding in the development of empirically testable hypotheses.

High-performing plastic clones best explain the spread of yellow monkeyflower from lowland to higher elevation areas in New Zealand.

The relative contribution of adaptation and phenotypic plasticity can vary between core and edge populations, with implications for invasive success. We investigated the spread of the invasive yellow monkeyflower, Erythranthe gutatta in New Zealand, where it is spreading from lowland agricultural land into high-elevation conservation areas. We investigated the extent of phenotypic variation among clones from across the South Island, looked for adaptation and compared degrees of plasticity among lowland core versus montane range-edge populations. We grew 34 clones and measured their vegetative and floral traits in two common gardens, one in the core range at 9 m a.s.l. and one near the range-edge at 560 m a.s.l. Observed trait variation was explained by a combination of genotypic diversity (as identified through common gardens) and high phenotypic plasticity. We found a subtle signature of local adaptation to lowland habitats but all clones were plastic and able to survive and reproduce in both gardens. In the range-edge garden, above-ground biomass was on average almost double and stolon length almost half that of the same clone in the core garden. Clones from low-elevation sites showed higher plasticity on average than those from higher elevation sites. The highest performing clones in the core garden were also top performers in the range-edge garden. These results suggest some highly fit general-purpose genotypes, possibly pre-adapted to New Zealand montane conditions, best explains the spread of E. gutatta from lowland to higher elevation areas.

Genomic mechanisms and consequences of diverse postzygotic barriers between monkeyflower species.

The evolution of genomic incompatibilities causing postzygotic barriers to hybridization is a key step in species divergence. Incompatibilities take 2 general forms-structural divergence between chromosomes leading to severe hybrid sterility in F1 hybrids and epistatic interactions between genes causing reduced fitness of hybrid gametes or zygotes (Dobzhansky-Muller incompatibilities). Despite substantial recent progress in understanding the molecular mechanisms and evolutionary origins of both types of incompatibility, how each behaves across multiple generations of hybridization remains relatively unexplored. Here, we use genetic mapping in F2 and recombinant inbred line (RIL) hybrid populations between the phenotypically divergent but naturally hybridizing monkeyflowers Mimulus cardinalis and M. parishii to characterize the genetic basis of hybrid incompatibility and examine its changing effects over multiple generations of experimental hybridization. In F2s, we found severe hybrid pollen inviability (<50% reduction vs parental genotypes) and pseudolinkage caused by a reciprocal translocation between Chromosomes 6 and 7 in the parental species. RILs retained excess heterozygosity around the translocation breakpoints, which caused substantial pollen inviability when interstitial crossovers had not created compatible heterokaryotypic configurations. Strong transmission ratio distortion and interchromosomal linkage disequilibrium in both F2s and RILs identified a novel 2-locus genic incompatibility causing sex-independent gametophytic (haploid) lethality. The latter interaction eliminated 3 of the expected 9 F2 genotypic classes via F1 gamete loss without detectable effects on the pollen number or viability of F2 double heterozygotes. Along with the mapping of numerous milder incompatibilities, these key findings illuminate the complex genetics of plant hybrid breakdown and are an important step toward understanding the genomic consequences of natural hybridization in this model system.

Unique drought resistance strategies occur among monkeyflower populations spanning an aridity gradient.

PREMISE: Annual plants often exhibit drought-escape and avoidance strategies to cope with limited water availability. Determining the extent of variation and factors underlying the evolution of divergent strategies is necessary for determining population responses to more frequent and severe droughts. METHODS: We leveraged five Mimulus guttatus populations collected across an aridity gradient within manipulative drought and quantitative genetics experiments to examine constitutive and terminal-drought induced responses in drought resistance traits. RESULTS: Populations varied considerably in drought-escape- and drought-avoidance-associated traits. The most mesic population demonstrated a unique resource conservative strategy. Xeric populations exhibited extreme plasticity when exposed to terminal drought that included flowering earlier at shorter heights, increasing water-use efficiency, and shifting C:N ratios. However, plasticity responses also differed between populations, with two populations slowing growth rates and flowering at earlier nodes and another population increasing growth rate. While nearly all traits were heritable, phenotypic correlations differed substantially between treatments and often, populations. CONCLUSIONS: Our results suggest drought resistance strategies of populations may be finely adapted to local patterns of water availability. Substantial plastic responses suggest that xeric populations can already acclimate to drought through plasticity, but populations not frequently exposed to drought may be more vulnerable.

Metriocnemus erythranthei sp. nov. and Limnophyes viribus sp. nov. (Diptera: Chironomidae: Orthocladiinae): leafminers of monkeyflowers, speedwells, and other herbaceous plants, with new observations on the ecology and habitats of other leaf-mining Chironomidae.

In this study, we describe Metriocnemus erythranthei sp. nov. and Limnophyes viribus sp. nov., leafminers of herbaceous wetland plants. The M. erythranthei larva is a true miner entering fresh leaves and excavating the tunnels, and the L. viribus larva inhabits vacated mines of M. erythranthei. M. erythranthei is widespread in North America, with collections from the Pacific coast to Pennsylvania, and L. viribus has been collected from Iowa and Oregon. We also describe the larva of a possible new species associated with these plants, which we refer to as Metriocnemus sp. "Oregon". A key to the known larval stages of North American Metriocnemus is also provided. Along with providing a detailed account of the mining ecology of these new species, we discuss additional observations of mostly Orthocladiinae midges associated with aquatic and terrestrial plants. These include documenting the rearing of Metriocnemus eurynotus (Holmgren, 1883) from larvae feeding on Impatiens (Balsaminaceae) cotyledons, initially as leafminers and later externally. Larvae of M. eurynotus also were found feeding within mines of M. erythranthei on Veronica (Plantaginaceae) and were collected along with M. erythranthei larvae on leaves of Petasites (Asteraceae).

Weak reproductive isolation and extensive gene flow between Mimulus glaucescens and M. guttatus in northern California.

Barriers to reproduction are often how progress in speciation is measured. Nonetheless, an unresolved question concerns the extent to which reproductive barriers diminish gene flow between incipient species. The Sierra Nevada foothill endemic Mimulus glaucescens and the widespread M. guttatus are considered distinct species based on striking differences in vegetative morphology, but barriers to reproduction have not been previously identified, nor has gene flow between species been characterized. Here, we examined 15 potential reproductive barriers within a Northern California area of broad sympatry. Most barriers, with the exception of ecogeographic isolation, were weak or absent, and total isolation for each species was incomplete. Population genomic analyses of range-wide and broadly sympatric accessions revealed extensive gene flow between these taxa, particularly in sympatry. Despite widespread introgression, Mimulus glaucescens, emerged as monophyletic and largely comprised a single ancestry that was found at intermediate frequency within M. guttatus. This result, along with observed ecological and phenotypic differentiation, suggests that natural selection may contribute to the maintenance of distinct phenotypic forms in the earliest stages of speciation. Integrating estimates of barrier strength with direct estimates of gene flow can strengthen a more nuanced interpretation of the process of speciation in natural communities.

Spatially and temporally varying selection influence species boundaries in two sympatric Mimulus.

Spatially and temporally varying selection can maintain genetic variation within and between populations, but it is less well known how these forces influence divergence between closely related species. We identify the interaction of temporal and spatial variation in selection and their role in either reinforcing or eroding divergence between two closely related Mimulus species. Using repeated reciprocal transplant experiments with advanced generation hybrids, we compare the strength of selection on quantitative traits involved in adaptation and reproductive isolation in Mimulus guttatus and Mimulus laciniatus between two years with dramatically different water availability. We found strong divergent habitat-mediated selection on traits in the direction of species differences during a drought in 2013, suggesting that spatially varying selection maintains species divergence. However, a relaxation in divergent selection on most traits in an unusually wet year (2019), including flowering time, which is involved in pre-zygotic isolation, suggests that temporal variation in selection may weaken species differences. Therefore, we find evidence that temporally and spatially varying selection may have opposing roles in mediating species boundaries. Given our changing climate, future growing seasons are expected to be more similar to the dry year, suggesting that in this system climate change may actually increase species divergence.

Taxon-specific, phased siRNAs underlie a speciation locus in monkeyflowers.

Taxon-specific small RNA loci are widespread in eukaryotic genomes, yet their role in lineage-specific adaptation, phenotypic diversification, and speciation is poorly understood. Here, we report that a speciation locus in monkeyflowers (Mimulus), YELLOW UPPER (YUP), contains an inverted repeat region that produces small interfering RNAs (siRNAs) in a phased pattern. Although the inverted repeat is derived from a partial duplication of a protein-coding gene that is not involved in flower pigmentation, one of the siRNAs targets and represses a master regulator of floral carotenoid pigmentation. YUP emerged with two protein-coding genes that control other aspects of flower coloration as a "superlocus" in a subclade of Mimulus and has contributed to subsequent phenotypic diversification and pollinator-mediated speciation in the descendant species.

Unusual suspects in flower color evolution.

The expression of phased small interfering RNAs caused monkeyflower color evolution.

Life history and chemical defense interact to drive patterns of adaptation in an annual monkeyflower.

Although chemical defenses and herbivore pressure are widely established as key targets and agents of selection, their roles in local adaptation and determining potential evolutionary responses to changing climates are often neglected. Here, we explore fitness differences between 11 rangewide M. guttatus populations in a field common garden experiment and assess the agents and targets of selection driving relative fitness patterns. We use piecewise structural equation models to disentangle associations between chemical defenses, (phenylpropanoid glycosides; PPGs), and life history traits with herbivory and fitness. While the historical environment of populations is not predictive of fitness differences between populations, >90% of variation in fitness can be predicted by the flowering time and foliar PPG defense arsenal of a population. Piecewise structural equation models indicate that life history traits, particularly earlier flowering time, are strongly and directly linked to fitness. However, herbivory, particularly fruit predation, is also an important agent of selection that creates indirect links between fitness and both chemical defenses and life history traits. Our results emphasize the multivariate nature of the agents and targets of selections in producing adaptation and suggest that future responses to selection must navigate a complex fitness landscape.

Integrating top-down and bottom-up approaches to understand the genetic architecture of speciation across a monkeyflower hybrid zone.

Understanding the phenotypic and genetic architecture of reproductive isolation is a long-standing goal of speciation research. In several systems, large-effect loci contributing to barrier phenotypes have been characterized, but such causal connections are rarely known for more complex genetic architectures. In this study, we combine "top-down" and "bottom-up" approaches with demographic modelling toward an integrated understanding of speciation across a monkeyflower hybrid zone. Previous work suggests that pollinator visitation acts as a primary barrier to gene flow between two divergent red- and yellow-flowered ecotypes of Mimulus aurantiacus. Several candidate isolating traits and anonymous single nucleotide polymorphism loci under divergent selection have been identified, but their genomic positions remain unknown. Here, we report findings from demographic analyses that indicate this hybrid zone formed by secondary contact, but that subsequent gene flow was restricted by widespread barrier loci across the genome. Using a novel, geographic cline-based genome scan, we demonstrate that candidate barrier loci are broadly distributed across the genome, rather than mapping to one or a few "islands of speciation." Quantitative trait locus (QTL) mapping reveals that most floral traits are highly polygenic, with little evidence that QTL colocalize, indicating that most traits are genetically independent. Finally, we find little evidence that QTL and candidate barrier loci overlap, suggesting that some loci contribute to other forms of reproductive isolation. Our findings highlight the challenges of understanding the genetic architecture of reproductive isolation and reveal that barriers to gene flow other than pollinator isolation may play an important role in this system.

The Climatic Variability Hypothesis and trade-offs in thermal performance in coastal and inland populations of Mimulus guttatus.

Ecologists and evolutionary biologists have long predicted that organisms in more climatically variable environments should be adapted to handle a wider range of conditions. This intuitive idea, known as the Climatic Variability Hypothesis (CVH), has gained mixed support from empirical studies. We tested the CVH in a novel system by comparing the thermal breadth of coastal and inland populations of Mimulus guttatus. To quantify thermal breadth, we performed a thermal performance experiment and built performance curves. Using these performance curves, we also evaluated evidence for a breadth-performance trade-off and the Hotter-is-Better hypothesis. We did not find support for the CVH; coastal and inland populations did not differ in thermal breadth. However, we found evidence for a breadth-performance trade-off and the Hotter-is-Better hypothesis. Surprisingly, the two most inland populations differed the most in the thermal performance traits we evaluated. Our results highlight the importance of explicitly measuring thermal performance to test explanations of species distribution patterns and the need to examine alternative mechanisms by which organisms occupy different climatic regimes.

To stripe or not to stripe: the origin of a novel foliar pigmentation pattern in monkeyflowers (Mimulus).

The origin of phenotypic novelty is one of the most challenging problems in evolutionary biology. Although genetic regulatory network rewiring or co-option has been widely recognised as a major contributor, in most cases how such genetic rewiring/co-option happens is completely unknown. We have studied a novel foliar pigmentation pattern that evolved recently in the monkeyflower species Mimulus verbenaceus. Through genome-wide association tests using wild-collected samples, experimental crosses of laboratory inbred lines, gene expression analyses, and functional assays, we identified an anthocyanin-activating R2R3-MYB gene, STRIPY, as the causal gene triggering the emergence of the discrete, mediolateral anthocyanin stripe in the M. verbenaceus leaf. Chemical mutagenesis revealed the existence of upstream activators and repressors that form a 'hidden' prepattern along the leaf proximodistal axis, potentiating the unique expression pattern of STRIPY. Population genomics analyses did not reveal signatures of positive selection, indicating that nonadaptive processes may be responsible for the establishment of this novel trait in the wild. This study demonstrates that the origin of phenotypic novelty requires at least two separate phases, potentiation and actualisation. The foliar stripe pattern of M. verbenaceus provides an excellent platform to probe the molecular details of both processes in future studies.

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.