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.

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.

Regional differences in leaf evolution facilitate photosynthesis following severe drought.

Characterizing physiological and anatomical changes that underlie rapid evolution following climatic perturbation can broaden our understanding of how climate change is affecting biodiversity. It can also provide evidence of cryptic adaptation despite stasis at higher levels of biological organization. Here, we compared evolutionary changes in populations of Mimulus cardinalis from historically different climates in the north and south of the species' range following an exceptional drought. We grew seeds produced from predrought ancestral plants alongside peak-drought descendants in a common glasshouse and exposed them to wet and dry conditions. Before the drought, northern ancestral populations expressed traits contributing to drought escape, while southern ancestral populations expressed drought avoidance. Following the drought, both regions evolved to reduce water loss and maintain photosynthesis in dry treatments (drought avoidance), but via different anatomical alterations in stomata, trichomes, and palisade mesophyll. Additionally, southern populations lost the ability to take advantage of wet conditions. These results reveal rapid evolution towards drought avoidance at an anatomical level following an exceptional drought, but suggest that differences in the mechanisms between regions incur different trade-offs. This sheds light on the importance of characterizing underlying mechanisms for downstream life-history and macromorphological traits.

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.

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).

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.

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.

Strong postmating reproductive isolation in Mimulus section Eunanus.

Postmating reproductive isolation can help maintain species boundaries when premating barriers to reproduction are incomplete. The strength and identity of postmating reproductive barriers are highly variable among diverging species, leading to questions about their genetic basis and evolutionary drivers. These questions have been tackled in model systems but are less often addressed with broader phylogenetic resolution. In this study we analyse patterns of genetic divergence alongside direct measures of postmating reproductive barriers in an overlooked group of sympatric species within the model monkeyflower genus, Mimulus. Within this Mimulus brevipes species group, we find substantial divergence among species, including a cryptic genetic lineage. However, rampant gene discordance and ancient signals of introgression suggest a complex history of divergence. In addition, we find multiple strong postmating barriers, including postmating prezygotic isolation, hybrid seed inviability and hybrid male sterility. M. brevipes and M. fremontii have substantial but incomplete postmating isolation. For all other tested species pairs, we find essentially complete postmating isolation. Hybrid seed inviability appears linked to differences in seed size, providing a window into possible developmental mechanisms underlying this reproductive barrier. While geographic proximity and incomplete mating isolation may have allowed gene flow within this group in the distant past, strong postmating reproductive barriers today have likely played a key role in preventing ongoing introgression. By producing foundational information about reproductive isolation and genomic divergence in this understudied group, we add new diversity and phylogenetic resolution to our understanding of the mechanisms of plant speciation.

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.

The Demographic Costs of Sexually Antagonistic Selection in Partially Selfing Populations.

AbstractWhen selection differs between the sexes, genes expressed by both males and females can experience sexually antagonistic (SA) selection, where beneficial alleles for one sex are deleterious for the other. Classic population genetics theory has been fundamental to understanding how and when SA genetic variation can be maintained by balancing selection, but these models have rarely considered the demographic consequences of coexisting alleles with deleterious fitness effects in each sex. In this article, we develop a stage-structured Mendelian matrix model and jointly analyze the evolutionary and demographic consequences of SA selection in obligately outcrossing (i.e., dioecious/gonochorous) and partially selfing hermaphrodite populations. We focus on identifying when SA polymorphisms are maintained by balancing selection and the population growth rate remains positive. Additionally, we analyze the effects of inbreeding depression manifesting at different life history stages and give an illustrative example of the potential for SA polymorphism in real populations using empirically estimated demographic rates for the hermaphroditic flowering plant Mimulus guttatus. Our results show that when population intrinsic growth rates approach 1, extinction occurs across large swathes of parameter space, favoring SA polymorphism or the fixation of male-beneficial alleles, and that inbreeding depression is a significant problem for maintaining SA polymorphism in partially selfing populations. Despite these demographic challenges, our example with M. guttatus appears to show that demographic rates observed in some real populations can sustain large regions of viable SA polymorphic space.

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.

Spatial variation in high temperature-regulated gene expression predicts evolution of plasticity with climate change in the scarlet monkeyflower.

A major way that organisms can adapt to changing environmental conditions is by evolving increased or decreased phenotypic plasticity. In the face of current global warming, more attention is being paid to the role of plasticity in maintaining fitness as abiotic conditions change over time. However, given that temporal data can be challenging to acquire, a major question is whether evolution in plasticity across space can predict adaptive plasticity across time. In growth chambers simulating two thermal regimes, we generated transcriptome data for western North American scarlet monkeyflowers (Mimulus cardinalis) collected from different latitudes and years (2010 and 2017) to test hypotheses about how plasticity in gene expression is responding to increases in temperature, and if this pattern is consistent across time and space. Supporting the genetic compensation hypothesis, individuals whose progenitors were collected from the warmer-origin northern 2017 descendant cohort showed lower thermal plasticity in gene expression than their cooler-origin northern 2010 ancestors. This was largely due to a change in response at the warmer (40°C) rather than cooler (20°C) treatment. A similar pattern of reduced plasticity, largely due to a change in response at 40°C, was also found for the cooler-origin northern versus the warmer-origin southern population from 2017. Our results demonstrate that reduced phenotypic plasticity can evolve with warming and that spatial and temporal changes in plasticity predict one another.

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.

Phylogenomic analyses in Phrymaceae reveal extensive gene tree discordance in relationships among major clades.

PREMISE: Phylogenomic datasets using genomes and transcriptomes provide rich opportunities beyond resolving bifurcating phylogenetic relationships. Monkeyflower (Phrymaceae) is a model system for evolutionary ecology. However, it lacks a well-supported phylogeny as a basis for a stable taxonomy and for macroevolutionary comparisons. METHODS: We sampled 24 genomes and transcriptomes in Phrymaceae and closely related families, including eight newly sequenced transcriptomes. We reconstructed the phylogeny using IQ-TREE and ASTRAL, evaluated gene tree discordance using PhyParts, Quartet Sampling, and a cloudogram, and carried out reticulation analyses using PhyloNet and HyDe. We searched for whole genome duplication (WGD) events using chromosome numbers, synonymous distances, and gene duplication events as evidence. RESULTS: Most gene trees support the monophyly of Phrymaceae and each of its tribes. Most gene trees also support tribe Mimuleae being sister to Phrymeae + Diplaceae + Leucocarpeae, with extensive gene tree discordance among the latter three. Despite the discordance, the monophyly of Mimulus s.l. is rejected, and no individual reticulation event among the Phrymaceae tribes is well-supported. Reticulation likely occurred among Erythranthe bicolor and closely related species. No ancient WGD was detected in Phrymaceae. Instead, small-scale duplications are among potential drivers of macroevolutionary diversification of Phrymaceae. CONCLUSIONS: We show that analysis of reticulate evolution is sensitive to taxon sampling and methods used. We also demonstrate that phylogenomic datasets using genomes and transcriptomes present rich opportunities to investigate gene family evolution and genome duplication events involved in lineage diversification and adaptation.

Rampant Misexpression in a Mimulus (Monkeyflower) Introgression Line Caused by Hybrid Sterility, Not Regulatory Divergence.

Divergence in gene expression regulation is common between closely related species and may give rise to incompatibilities in their hybrid progeny. In this study, we investigated the relationship between regulatory evolution within species and reproductive isolation between species. We focused on a well-studied case of hybrid sterility between two closely related yellow monkeyflower species, Mimulus guttatus and Mimulus nasutus, that is caused by two epistatic loci, hybrid male sterility 1 (hms1) and hybrid male sterility 2 (hms2). We compared genome-wide transcript abundance across male and female reproductive tissues (i.e., stamens and carpels) from four genotypes: M. guttatus, M. nasutus, and sterile and fertile progeny from an advanced M. nasutus-M. guttatus introgression line carrying the hms1-hms2 incompatibility. We observed substantial variation in transcript abundance between M. guttatus and M. nasutus, including distinct but overlapping patterns of tissue-biased expression, providing evidence for regulatory divergence between these species. We also found rampant genome-wide misexpression, but only in the affected tissues (i.e., stamens) of sterile introgression hybrids carrying incompatible alleles at hms1 and hms2. Examining patterns of allele-specific expression in sterile and fertile introgression hybrids, we found evidence for interspecific divergence in cis- and trans-regulation, including compensatory cis-trans mutations likely to be driven by stabilizing selection. Nevertheless, species divergence in gene regulatory networks cannot explain the vast majority of the gene misexpression we observe in Mimulus introgression hybrids, which instead likely manifests as a downstream consequence of sterility itself.