The quantitative genetics of gene expression in Mimulus guttatus.

Gene expression can be influenced by genetic variants that are closely linked to the expressed gene (cis eQTLs) and variants in other parts of the genome (trans eQTLs). We created a multiparental mapping population by sampling genotypes from a single natural population of Mimulus guttatus and scored gene expression in the leaves of 1,588 plants. We find that nearly every measured gene exhibits cis regulatory variation (91% have FDR < 0.05). cis eQTLs are usually allelic series with three or more functionally distinct alleles. The cis locus explains about two thirds of the standing genetic variance (on average) but varies among genes and tends to be greatest when there is high indel variation in the upstream regulatory region and high nucleotide diversity in the coding sequence. Despite mapping over 10,000 trans eQTL / affected gene pairs, most of the genetic variance generated by trans acting loci remains unexplained. This implies a large reservoir of trans acting genes with subtle or diffuse effects. Mapped trans eQTLs show lower allelic diversity but much higher genetic dominance than cis eQTLs. Several analyses also indicate that trans eQTLs make a substantial contribution to the genetic correlations in expression among different genes. They may thus be essential determinants of "gene expression modules," which has important implications for the evolution of gene expression and how it is studied by geneticists.

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.

Weak response to selection on stigma-anther distance in a primarily selfing population of yellow monkeyflower.

Stebbins hypothesized that selfing lineages are evolutionary dead ends because they lack adaptive potential. While selfing populations often possess limited nucleotide variability compared with closely related outcrossers, reductions in the genetic variability of quantitative characters remain unclear, especially for key traits determining selfing rates. Yellow monkeyflower (Mimulus guttatus) populations generally outcross and maintain extensive quantitative genetic variation in floral traits. Here, we study the Joy Road population (Bodega Bay, CA, USA) of M. guttatus, where individuals exhibit stigma-anther distances (SAD) typical of primarily selfing monkeyflowers. We show that this population is closely related to nearby conspecifics on the Pacific Coast with a modest 33% reduction in genome-wide variation compared with a more highly outcrossing population. A five-generation artificial selection experiment challenged the hypothesis that the Joy Road population harbours comparatively low evolutionary potential in stigma-anther distance, a critical determinant of selfing rate in Mimulus. Artificial selection generated a weak phenotypic response, with low realized heritabilities (0.020-0.028) falling 84% below those measured for floral characters in more highly outcrossing M. guttatus. These results demonstrate substantial declines in evolutionary potential with a transition toward selfing. Whether these findings explain infrequent reversals to outcrossing or general limits on adaptation in selfers requires further investigation.

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.

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.

Endosperm-based incompatibilities in hybrid monkeyflowers.

Endosperm is an angiosperm innovation central to their reproduction whose development, and thus seed viability, is controlled by genomic imprinting, where expression from certain genes is parent-specific. Unsuccessful imprinting has been linked to failed inter-specific and inter-ploidy hybridization. Despite their importance in plant speciation, the underlying mechanisms behind these endosperm-based barriers remain poorly understood. Here, we describe one such barrier between diploid Mimulus guttatus and tetraploid Mimulus luteus. The two parents differ in endosperm DNA methylation, expression dynamics, and imprinted genes. Hybrid seeds suffer from underdeveloped endosperm, reducing viability, or arrested endosperm and seed abortion when M. guttatus or M. luteus is seed parent, respectively, and transgressive methylation and expression patterns emerge. The two inherited M. luteus subgenomes, genetically distinct but epigenetically similar, are expressionally dominant over the M. guttatus genome in hybrid embryos and especially their endosperm, where paternal imprints are perturbed. In aborted seeds, de novo methylation is inhibited, potentially owing to incompatible paternal instructions of imbalanced dosage from M. guttatus imprints. We suggest that diverged epigenetic/regulatory landscapes between parental genomes induce epigenetic repatterning and global shifts in expression, which, in endosperm, may uniquely facilitate incompatible interactions between divergent imprinting schemes, potentially driving rapid barriers.

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).

Predicting evolutionary change at the DNA level in a natural Mimulus population.

Evolution by natural selection occurs when the frequencies of genetic variants change because individuals differ in Darwinian fitness components such as survival or reproductive success. Differential fitness has been demonstrated in field studies of many organisms, but it remains unclear how well we can quantitatively predict allele frequency changes from fitness measurements. Here, we characterize natural selection on millions of Single Nucleotide Polymorphisms (SNPs) across the genome of the annual plant Mimulus guttatus. We use fitness estimates to calibrate population genetic models that effectively predict allele frequency changes into the next generation. Hundreds of SNPs experienced "male selection" in 2013 with one allele at each SNP elevated in frequency among successful male gametes relative to the entire population of adults. In the following generation, allele frequencies at these SNPs consistently shifted in the predicted direction. A second year of study revealed that SNPs had effects on both viability and reproductive success with pervasive trade-offs between fitness components. SNPs favored by male selection were, on average, detrimental to survival. These trade-offs (antagonistic pleiotropy and temporal fluctuations in fitness) may be essential to the long-term maintenance of alleles. Despite the challenges of measuring selection in the wild, the strong correlation between predicted and observed allele frequency changes suggests that population genetic models have a much greater role to play in forward-time prediction of evolutionary change.

Selfish chromosomal drive shapes recent centromeric histone evolution in monkeyflowers.

Centromeres are essential mediators of chromosomal segregation, but both centromeric DNA sequences and associated kinetochore proteins are paradoxically diverse across species. The selfish centromere model explains rapid evolution by both components via an arms-race scenario: centromeric DNA variants drive by distorting chromosomal transmission in female meiosis and attendant fitness costs select on interacting proteins to restore Mendelian inheritance. Although it is clear than centromeres can drive and that drive often carries costs, female meiotic drive has not been directly linked to selection on kinetochore proteins in any natural system. Here, we test the selfish model of centromere evolution in a yellow monkeyflower (Mimulus guttatus) population polymorphic for a costly driving centromere (D). We show that the D haplotype is structurally and genetically distinct and swept to a high stable frequency within the past 1500 years. We use quantitative genetic mapping to demonstrate that context-dependence in the strength of drive (from near-100% D transmission in interspecific hybrids to near-Mendelian in within-population crosses) primarily reflects variable vulnerability of the non-driving competitor chromosomes, but also map an unlinked modifier of drive coincident with kinetochore protein Centromere-specific Histone 3 A (CenH3A). Finally, CenH3A exhibits a recent (<1000 years) selective sweep in our focal population, implicating local interactions with D in ongoing adaptive evolution of this kinetochore protein. Together, our results demonstrate an active co-evolutionary arms race between DNA and protein components of the meiotic machinery in Mimulus, with important consequences for individual fitness and molecular divergence.

Ancient and recent introgression shape the evolutionary history of pollinator adaptation and speciation in a model monkeyflower radiation (Mimulus section Erythranthe).

Inferences about past processes of adaptation and speciation require a gene-scale and genome-wide understanding of the evolutionary history of diverging taxa. In this study, we use genome-wide capture of nuclear gene sequences, plus skimming of organellar sequences, to investigate the phylogenomics of monkeyflowers in Mimulus section Erythranthe (27 accessions from seven species). Taxa within Erythranthe, particularly the parapatric and putatively sister species M. lewisii (bee-pollinated) and M. cardinalis (hummingbird-pollinated), have been a model system for investigating the ecological genetics of speciation and adaptation for over five decades. Across >8000 nuclear loci, multiple methods resolve a predominant species tree in which M. cardinalis groups with other hummingbird-pollinated taxa (37% of gene trees), rather than being sister to M. lewisii (32% of gene trees). We independently corroborate a single evolution of hummingbird pollination syndrome in Erythranthe by demonstrating functional redundancy in genetic complementation tests of floral traits in hybrids; together, these analyses overturn a textbook case of pollination-syndrome convergence. Strong asymmetries in allele sharing (Patterson's D-statistic and related tests) indicate that gene tree discordance reflects ancient and recent introgression rather than incomplete lineage sorting. Consistent with abundant introgression blurring the history of divergence, low-recombination and adaptation-associated regions support the new species tree, while high-recombination regions generate phylogenetic evidence for sister status for M. lewisii and M. cardinalis. Population-level sampling of core taxa also revealed two instances of chloroplast capture, with Sierran M. lewisii and Southern Californian M. parishii each carrying organelle genomes nested within respective sympatric M. cardinalis clades. A recent organellar transfer from M. cardinalis, an outcrosser where selfish cytonuclear dynamics are more likely, may account for the unexpected cytoplasmic male sterility effects of selfer M. parishii organelles in hybrids with M. lewisii. Overall, our phylogenomic results reveal extensive reticulation throughout the evolutionary history of a classic monkeyflower radiation, suggesting that natural selection (re-)assembles and maintains species-diagnostic traits and barriers in the face of gene flow. Our findings further underline the challenges, even in reproductively isolated species, in distinguishing re-use of adaptive alleles from true convergence and emphasize the value of a phylogenomic framework for reconstructing the evolutionary genetics of adaptation and speciation.

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.

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.

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.

Developmental Genetics of Corolla Tube Formation: Role of the tasiRNA-ARF Pathway and a Conceptual Model.

Over 80,000 angiosperm species produce flowers with petals fused into a corolla tube. The corolla tube contributes to the tremendous diversity of flower morphology and plays a critical role in plant reproduction, yet it remains one of the least understood plant structures from a developmental genetics perspective. Through mutant analyses and transgenic experiments, we show that the tasiRNA-ARF pathway is required for corolla tube formation in the monkeyflower species Mimulus lewisii Loss-of-function mutations in the M. lewisii orthologs of ARGONAUTE7 and SUPPRESSOR OF GENE SILENCING3 cause a dramatic decrease in abundance of TAS3-derived small RNAs and a moderate upregulation of AUXIN RESPONSE FACTOR3 (ARF3) and ARF4, which lead to inhibition of lateral expansion of the bases of petal primordia and complete arrest of the upward growth of the interprimordial regions, resulting in unfused corollas. Using the DR5 auxin-responsive promoter, we discovered that auxin signaling is continuous along the petal primordium base and the interprimordial region during the critical stage of corolla tube formation in the wild type, similar to the spatial pattern of MlARF4 expression. Auxin response is much weaker and more restricted in the mutant. Furthermore, exogenous application of a polar auxin transport inhibitor to wild-type floral apices disrupted petal fusion. Together, these results suggest a new conceptual model highlighting the central role of auxin-directed synchronized growth of the petal primordium base and the interprimordial region in corolla tube formation.

A Tetratricopeptide Repeat Protein Regulates Carotenoid Biosynthesis and Chromoplast Development in Monkeyflowers (Mimulus).

Little is known about the factors regulating carotenoid biosynthesis in flowers. Here, we characterized the REDUCED CAROTENOID PIGMENTATION2 (RCP2) locus from two monkeyflower (Mimulus) species, the bumblebee-pollinated species Mimulus lewisii and the hummingbird-pollinated species Mimulus verbenaceus We show that loss-of-function mutations of RCP2 cause drastic down-regulation of the entire carotenoid biosynthetic pathway. The causal gene underlying RCP2 encodes a tetratricopeptide repeat protein that is closely related to the Arabidopsis (Arabidopsis thaliana) REDUCED CHLOROPLAST COVERAGE proteins. RCP2 appears to regulate carotenoid biosynthesis independently of RCP1, a previously identified R2R3-MYB master regulator of carotenoid biosynthesis. We show that RCP2 is necessary and sufficient for chromoplast development and carotenoid accumulation in floral tissues. Simultaneous down-regulation of RCP2 and two closely related paralogs, RCP2-L1 and RCP2-L2, yielded plants with pale leaves deficient in chlorophyll and carotenoids and with reduced chloroplast compartment size. Finally, we demonstrate that M. verbenaceus is just as amenable to chemical mutagenesis and in planta transformation as the more extensively studied M. lewisii, making these two species an excellent platform for comparative developmental genetics studies of closely related species with dramatic phenotypic divergence.

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.

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.

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.

Frequency-Dependent Hybridization Contributes to Habitat Segregation in Monkeyflowers.

AbstractSpatial segregation of closely related species is usually attributed to differences in stress tolerance and competitive ability. For both animals and plants, reproductive interactions between close relatives can impose a fitness cost that is more detrimental to the rarer species. Frequency-dependent mating interactions may thus prevent the establishment of immigrants within heterospecific populations, maintaining spatial segregation of species. Despite strong spatial segregation in natural populations, two sympatric California monkeyflowers (Mimulus nudatus and M. guttatus) survive and reproduce in the other's habitat when transplanted reciprocally. We hypothesized that a frequency-dependent mating disadvantage maintains spatial segregation of these monkeyflowers during natural immigration. To evaluate this hypothesis, we performed two field experiments. First, we experimentally added immigrants in varying numbers to sites dominated by heterospecifics. Second, we reciprocally transplanted arrays of varying resident and immigrant frequencies. Immigrant seed viability decreased with conspecific rarity for M. guttatus but not for M. nudatus. We observed immigrant minority disadvantage for both species, but it was driven by different factors-frequency-dependent hybridization for M. guttatus and competition for resources and/or pollinators for M. nudatus. Overall, our results suggest a major role for reproductive interference in spatial segregation that should be evaluated along with stress tolerance and competitive ability.

Developmental evidence for parental conflict in driving Mimulus species barriers.

The endosperm, a tissue that nourishes the embryo in the seeds of flowering plants, is often disrupted in inviable hybrid seeds of closely related species. A key question is whether parental conflict is a major driver of this common form of reproductive isolation. Here, we performed reciprocal crosses between pairs of three monkeyflower species (Mimulus caespitosa, Mimulus tilingii, and Mimulus guttatus). The severity of hybrid seed inviability varies among these crosses, which we inferred to be due to species divergence in effective ploidy. By performing a time series experiment of seed development, we discovered parent-of-origin phenotypes that provide strong evidence for parental conflict in shaping endosperm evolution. We found that the chalazal haustorium, a tissue within the endosperm that is found at the maternal-filial boundary, shows pronounced differences between reciprocal hybrid seeds formed from Mimulus species that differ in effective ploidy. These parent-of-origin effects suggest that the chalazal haustorium might act as a mediator of parental conflict, potentially by controlling sucrose movement from the maternal parent into the endosperm. Our study suggests that parental conflict in the endosperm may function as a driver of speciation by targeting regions and developmental stages critical for resource allocation and thus proper seed development.