Loci affecting long-term hybrid survivorship in Louisiana irises: implications for reproductive isolation and introgression

Iris fulva and I. brevicaulis are long-lived plant species known to hybridize where they coexist in nature. Year-to-year survival contributes significantly to overall fitness for both species and their hybrid derivatives, and differences in hybrid survivability may have important consequences to interspecific gene flow in nature. We examined the genetic architecture of long-term survivorship of reciprocal backcross I. fulva x I. brevicaulis hybrids in a common-garden, greenhouse environment. Differences in mortality were found between the two backcross (BC1) hybrid classes, with hybrids crossed toward I. fulva (BCIF) revealing twice the mortality of those hybrids backcrossed toward I. brevicaulis (BCIB). Using genomic scans on two separate genetic linkage maps derived from the reciprocal hybrid populations, we found that hybrid survivorship is influenced by several genetic regions. Multiple interval mapping (MIM) revealed four quantitative trait loci (QTLs) in BCIF hybrids that were significantly associated with survivorship. Introgressed I. brevicaulis DNA increased survivorship at three of the four QTLs. For the fourth QTL, introgressed I. brevicaulis DNA was associated with decreased survivorship. No QTLs were detected in BCIB hybrids; however, single-marker analysis revealed five unlinked loci that were significantly associated with survivorship. At all five markers, survivorship was positively associated with introgressed I. fulva DNA. The present findings have important implications for the evolutionary dynamics of naturally occurring hybrid zones. Regions of the genome that increase survivorship when in a heterozygous (i.e., hybrid) state should have an increased likelihood of passing across species boundaries, whereas those that decrease survivorship will be less likely to introgress.     

The genetic architecture of reproductive isolation in Louisiana irises: flowering phenology

Despite the potential importance of divergent reproductive phenologies as a barrier to gene flow, we know less about the genetics of this factor than we do about any other isolating barrier. Here, we report on the genetic architecture of divergent flowering phenologies that result in substantial reproductive isolation between the naturally hybridizing plant species Iris fulva and I. brevicaulis. I. fulva initiates and terminates flowering significantly earlier than I. brevicaulis. We examined line crosses of reciprocal F1 and backcross (BC1) hybrids and determined that flowering time was polygenic in nature. We further defined quantitative trait loci (QTL) that affect the initiation of flowering in each of these species. QTL analyses were performed separately for two different growing seasons in the greenhouse, as well as in two field plots where experimental plants were placed into nature. For BCIF hybrids (BC1 toward I. fulva), 14 of 17 detected QTL caused flowering to occur later in the season when I. brevicaulis alleles were present, while the remaining 3 caused flowering to occur earlier. In BCIB hybrids (BC1 toward I. brevicaulis), 11 of 15 detected QTL caused flowering to occur earlier in the season when introgressed I. fulva alleles were present, while the remaining 4 caused flowering to occur later. These ratios are consistent with expectations of selection (as opposed to drift) promoting flowering divergence in the evolutionary history of these species. Furthermore, epistatic interactions among the QTL also reflected the same trends, with the majority of epistatic effects causing later flowering than expected in BCIF hybrids and earlier flowering in BCIB hybrids. Overlapping QTL that influenced flowering time across all four habitat/treatment types were not detected, indicating that increasing the sample size of genotyped plants would likely increase the number of significant QTL found in this study.     

The genetic architecture of reproductive isolation in Louisiana irises: pollination syndromes and pollinator preferences

In animal-pollinated plants, pollinator preferences for divergent floral forms can lead to partial reproductive isolation. We describe regions of plant genomes that affect pollinator preferences for two species of Louisiana Irises, Iris brevicaulis and Iris fulva, and their artificial hybrids. Iris brevicaulis and I. fulva possess bee and bird-pollination syndromes, respectively. Hummingbirds preferred I. fulva and under-visited both I. brevicaulis and backcrosses toward this species. Lepidopterans preferred I. fulva and backcrosses toward I. fulva, but also under-visited I. brevicaulis and I. brevicaulis backcrosses. Bumblebees preferred I. brevicaulis and F1 hybrids and rarely visited I. fulva. Although all three pollen vectors preferred one or the other species, these preferences did not prevent visitation to other hybrid/parental classes. Quantitative trait locus (QTL) mapping, in reciprocal BC1 mapping populations, defined the genetic architecture of loci that affected pollinator behavior. We detected six and nine QTLs that affected pollinator visitation rates in the BCIb and BCIf mapping populations, respectively, with as many as three QTLs detected for each trait. Overall, this study reflects the possible role of quantitative genetic factors in determining (1) reproductive isolation, (2) the pattern of pollinator-mediated genetic exchange, and thus (3) hybrid zone evolution.     

Genetic mapping of species boundaries in Louisiana irises using IRRE retrotransposon display markers

Genetic mapping studies provide insight into the pattern and extent of genetic incompatibilities affecting hybridization between closely related species. Genetic maps of two species of Louisiana Irises, Iris fulva and I. brevicaulis, were constructed from transposon-based molecular markers segregating in reciprocal backcross (BC1) interspecific hybrids and used to investigate genomic patterns of species barriers inhibiting introgression. Linkage mapping analyses indicated very little genetic incompatibility between I. fulva and I. brevicaulis in the form of map regions exhibiting transmission ratio distortion, and this was confirmed using a Bayesian multipoint mapping analysis. These results demonstrate the utility of transposon-based marker systems for genetic mapping studies of wild plant species and indicate that the genomes of I. fulva and I. brevicaulis are highly permeable to gene flow and introgression from one another via backcrossing.     

Environment-dependent performance and fitness of Iris brevicaulis, I. fulva (Iridaceae), and hybrids

We tested the relative fitness of two Louisiana Iris species (Iris brevicaulis and I. fulva) and their first-generation backcross hybrids in three experimental watering treatments: dry, field capacity, and flooded. Leaf area expansion rate, gas exchange (A(max), g(s), c(i)), and biomass at final harvest were measured for each species and hybrid class in all three environmental treatments. Fitness (based on total biomass) of the four genotypic classes differed significantly with environment. All genotypic classes performed most poorly in the dry treatment. The fitness ranking of genotypic class also changed across environments (significant genotypic class by treatment interaction) with hybrid genotype fitness shifting relative to parental genotypes. Integrating over all treatments, backcrosses to I. fulva showed the lowest fitness, whereas backcrosses to I. brevicaulis outperformed I. fulva. The differences in fitness were apparently achieved by a combination of differences in photosynthesis and allocation. In this system, hybrids are not necessarily less fit than their parents, and the relationship between hybrid and parental fitness is influenced by environmental conditions, lending support to the Hybrid Novelty model of hybrid zone evolution.     

THE GENETIC ARCHITECTURE OF REPRODUCTIVE ISOLATION IN LOUISIANA IRISES: HYBRID FITNESS IN NATURE

Negative epistasis in hybrid genomes commonly results in postzygotic isolation between divergent lineages. However, some genomic regions may be selectively neutral or adaptive in hybrids and thus may potentially cross species barriers. We examined postzygotic isolation between ecologically similar species of Louisiana Iris: Iris brevicaulis and I. fulva to determine the potential for adaptive introgression in nature. Line-cross analyses allowed us a general overview of the gene action responsible for fitness-related traits. We then used a QTL mapping approach to detect genomic regions responsible for variation in these traits. Although hybrid classes suffered reduced fitness for many traits, hybrid means were equivalent to at least one of the parental species in overall estimates of maternal and paternal fitness during the two years of the field study. The genetic architecture underlying the fitness-related traits varied across field site and year of the study, thus emphasizing the importance of the environment in determining the degree of postzygotic isolation and potential for introgression across natural hybrid zones.     

Nectar Production in Louisiana Iris Hybrids

Nectar is an important attractant for pollinators, and a plant's success in sexual reproduction can be influenced by the amount and concentration of nectar produced by its flowers. We studied nectar production over flower lifetime in Iris fulva, Iris brevicaulis, and four classes of hybrids-reciprocal F1's and backcrosses-between these species. Iris fulva produced less concentrated nectar than did I. brevicaulis, whereas I. brevicaulis flowers had a shorter life span. Hybrids were not intermediate, but they had the high nectar concentration of I. brevicaulis combined with the long life span of I. fulva flowers. Nectar production and concentration declined after the first day in all classes, but flowers continued to produce nectar until they were completely wilted. Backcrosses did not show a shift in mean or increased variation for the characters that distinguished the parental species; backcrosses toward I. fulva retained the high nectar concentration of I. brevicaulis, and backcrosses toward I. brevicaulis did not have a reduced flower life span. Overall, F1 hybrid flowers produced the highest amounts of nectar and nectar sugar over their life spans. These results, together with previously obtained data on pollinator choice in mixed arrays of the same flower classes, show that F1 hybrids between these species do not suffer from reduced attractiveness to pollinators. F1 individuals produced more nectar and nectar sugar than did their parents, and thus, they are possibly even more attractive to pollinators that forage for nectar.     

QTL mapping reveals the genetic architecture of loci affecting pre- and post-zygotic isolating barriers in Louisiana Iris

ABSTRACT: BACKGROUND: Hybridization among Louisiana Irises has been well established and the genetic architecture of reproductive isolation is known to affect the potential for and the directionality of introgression between taxa. Here we use co-dominant markers to identify regions where QTL are located both within and between backcross maps to compare the genetic architecture of reproductive isolation and fitness traits across treatments and years. RESULTS: QTL mapping was used to elucidate the genetic architecture of reproductive isolation between Iris fulva and Iris brevicaulis. Homologous co-dominant EST-SSR markers scored in two backcross populations between I. fulva and I. brevicaulis were used to generate genetic linkage maps. These were used as the framework for mapping QTL associated with variation in 11 phenotypic traits likely responsible for reproductive isolation and fitness. QTL were dispersed throughout the genome, with the exception of one region of a single linkage group (LG) where QTL for flowering time, sterility, and fruit production clustered. In most cases, homologous QTL were not identified in both backcross populations, however, homologous QTL for flowering time, number of growth points per rhizome, number of nodes per inflorescence, and number of flowers per node were identified on several linkage groups. CONCLUSION: Two different traits affecting reproductive isolation, flowering time and sterility, exhibit different genetic architectures, with numerous QTL across the Iris genome controlling flowering time and fewer, less distributed QTL affecting sterility. QTL for traits affecting fitness are largely distributed across the genome with occasional overlap, especially on LG 4, where several QTL increasing fitness and decreasing sterility cluster. Given the distribution and effect direction of QTL affecting reproductive isolation and fitness, we have predicted genomic regions where introgression may be more likely to occur (those regions associated with an increase in fitness and unlinked to loci controlling reproductive isolation) and those that are less likely to exhibit introgression (those regions linked to traits decreasing fitness and reproductive isolation).     

Intimately linked or hardly speaking? The relationship between genotype and environmental gradients in a Louisiana Iris hybrid population

Several models of hybrid zone evolution predict the same spatial patterns of genotypic distribution whether or not structuring is due to environment-dependent or -independent selection. In this study, we tested for evidence of environment-dependent selection in an Iris fulva x Iris brevicaulis hybrid population by examining the distribution of genotypes in relation to environmental gradients. We selected 201 Louisiana Iris plants from within a known hybrid population (80 m x 80 m) and placed them in four different genotypic classes (I. fulva, I. fulva-like hybrid, I. brevicaulis-like hybrid and I. brevicaulis) based on seven species-specific random amplified polymorphic DNA (RAPD) markers and two chloroplast DNA haplotypes. Environmental variables were then measured. These variables included percentage cover by tree canopy, elevation from the high water mark, soil pH and percentage soil organic matter. Each variable was sampled for all 201 plants. Canonical discriminant analysis (CDA) was used to infer the environmental factors most strongly associated with the different genotypic groups. Slight differences in elevation (-0.5 m to +0.4 m) were important for distinguishing habitat distributions described by CDA, even though there were no statistical differences between mean elevations alone. I. brevicaulis occurred in a broad range of habitats, while I. fulva had a narrower distribution. Of all the possible combinations, I. fulva-like hybrids and I. brevicaulis-like hybrids occurred in the most distinct habitat types relative to one another. Each hybrid class was not significantly different from its closest parent with regard to habitat occupied, but was statistically unique from its more distant parental species. Within the hybrid genotypes, most, but not all, RAPD loci were individually correlated with environmental variables. This study suggests that, at a very fine spatial scale, environment-dependent selection contributed to the genetic structuring of this hybrid zone.     

QTL analysis of floral traits in Louisiana iris hybrids

The formation of hybrid zones between nascent species is a widespread phenomenon. The evolutionary consequences of hybridization are influenced by numerous factors, including the action of natural selection on quantitative trait variation. Here we examine how the genetic basis of floral traits of two species of Louisiana Irises affects the extent of quantitative trait variation in their hybrids. Quantitative trait locus (QTL) mapping was used to assess the size (magnitude) of phenotypic effects of individual QTL, the degree to which QTL for different floral traits are colocalized, and the occurrence of mixed QTL effects. These aspects of quantitative genetic variation would be expected to influence (1) the number of genetic steps (in terms of QTL substitutions) separating the parental species phenotypes; (2) trait correlations; and (3) the potential for transgressive segregation in hybrid populations. Results indicate that some Louisiana Iris floral trait QTL have large effects and QTL for different traits tend to colocalize. Transgressive variation was observed for six of nine traits, despite the fact that mixed QTL effects influence few traits. Overall, our QTL results imply that the genetic basis of floral morphology and color traits might facilitate the maintenance of phenotypic divergence between Iris fulva and Iris brevicaulis, although a great deal of phenotypic variation was observed among hybrids.     

SHADE TOLERANCE AND ITS EFFECT ON THE SEGREGATION OF TWO SPECIES OF LOUISIANA IRIS AND THEIR HYBRIDS

Iris fulva Ker. Gawler and Iris hexagona Walter have overlapping geographic ranges in Louisiana. In areas of overlap hybrids are fairly common. Iris hexagona occupies the borders of freshwater marshes of southern Louisiana while I. fulva can be found farther north along edges of natural levees, canals and swamps. Where the natural levee penetrates the marsh, natural hybridization can occur between I. hexagona and I. fulva. It has been suggested that one principal explanation for the segregation of the two species is that I. fulva grows best in semishade and I. hexagona grows best in full sun. A greenhouse study was conducted using rhizomes collected from the field to test this hypothesis and determine the relative shade tolerance of two natural hybrid types. Iris fulva, I. hexagona, and the two hybrid taxa were grown under 0% (control), 50% (medium shade), and 80% (high shade) reduction of sunlight for 6 months and then harvested. Iris fulva was found to be more tolerant of shading than I. hexagona and the two hybrids. Further, I. fulva was found to grow as well in control as in medium shade. Both hybrid taxa were more shade tolerant than I. hexagona. Iris hexagona was greatly affected by all levels of shade. In general, the results suggest that these hybrids are intermediate to the parental taxa in terms of shade tolerance.     

Integrating Bayesian genomic cline analyses and association mapping of morphological and ecological traits to dissect reproductive isolation and introgression in a Louisiana Iris hybrid zone

Hybrid zones provide unique opportunities to examine reproductive isolation and introgression in nature. We utilized 45,384 single nucleotide polymorphism (SNP) loci to perform association mapping of 14 floral, vegetative and ecological traits that differ between Iris hexagona and Iris fulva, and to investigate, using a Bayesian genomic cline (BGC) framework, patterns of genomic introgression in a large and phenotypically diverse hybrid zone in southern Louisiana. Many loci of small effect size were consistently found to be associated with phenotypic variation across all traits, and several individual loci were revealed to influence phenotypic variation across multiple traits. Patterns of genomic introgression were quite heterogeneous throughout the Louisiana Iris genome, with I. hexagona alleles tending to be favoured over those of I. fulva. Loci that were found to have exceptional patterns of introgression were also found to be significantly associated with phenotypic variation in a small number of morphological traits. However, this was the exception rather than the rule, as most loci that were associated with morphological trait variation were not significantly associated with excess ancestry. These findings provide insights into the complexity of the genomic architecture of phenotypic differences and are a first step towards identifying loci that are associated with both trait variation and reproductive isolation in nature.     

Hybrid bridges to gene flow: a case study in milkweeds (Asclepias)

Natural hybridization occurs throughout areas of sympatry for the North American milkweeds Asclepias exaltata and A. syriaca (Asclepiadaceae), even though the formation of F1 hybrid seed is a rare event. For introgressive hybridization to proceed, F1 and advanced hybrids must be released from reproductive barriers and successfully mate with one or both parental species. I investigated the mating system of natural hybrids between A. exaltata and A. syriaca in three populations in Shenandoah National Park, Virginia. Allozyme data and a maximum-likelihood procedure were used to estimate the frequency of six genotypic classes (parentals, F1, F2, and backcrosses) of the hybridizing populations, the pollinia received by hybrid plants, and the paternal parents of seeds produced by hybrids. F1 hybrids, backcross A. syriaca, and parental A. syriaca individuals were common in three hybrid populations. Even though self-pollinations and interhybrid pollinations were common, F2 seed production and the occurrence of F2 individuals were rare in hybrid populations. Hybrid plants received more pollen from A. syriaca than A. exaltata, which resulted in the production of more backcross-A. syriaca seed than backcross-A. exaltata seed. Asclepias exaltata was rare in the hybrid populations, but A. exaltata pollinia were received by hybrids and this species sired between 15% and 36% of the seeds produced on hybrids. The potential for introgression with A. exaltata populations is lower because this species is unsuccessful as the maternal parent in interspecific and backcross hand-pollinations. The asymetry of hybridization with A. syriaca as the maternal parent is further supported by the incorporation of maternally inherited chloroplast DNA markers in hybrids. Hybrid milkweeds frequently backcross with both parental species and may be released from the reproductive barriers that limit the formation of F1 hybrids in natural populations. The direction of interspecific gene flow and introgression in milkweeds is influenced by the reproductive biology of hybrids, the constituency of the surrounding population, and failure of some crosses to produce seeds. Finally, introgressive hybridization remains an important evolutionary force even when the initial formation of F1 hybrids in natural populations is rare.     

HYBRID FITNESS IN THE LOUISIANA IRISES: ANALYSIS OF PARENTAL AND F1 PERFORMANCE

The assumption of hybrid inferiority is central to the two models most widely applied to the prediction of hybrid zone evolution. Both the tension zone and mosaic models assume that natural selection acts against hybrids regardless of the environment in which they occur. To test this assumption, we investigated components of fitness in Iris fulva, I. hexagona and their reciprocal F₁ hybrids under greenhouse conditions. The four cross types were compared on the basis of seed germination, vegetative and clonal growth, and sexual reproduction. In all cases, the hybrids performed as well as, or significantly better than, both of their parents. These results suggest that F₁ hybrids between I. fulva and I. hexagona are at least as fit as their parents. The results of this study are therefore inconsistent with the assumptions of both the tension zone and mosaic models of hybrid zone evolution.     

Gene flow from wheat (Triticum aestivum L.) to jointed goatgrass (Aegilops cylindrica Host.), as revealed by RAPD and microsatellite markers

In order to estimate the potential of gene flow between wheat (Triticum æstivum L.) and jointed goatgrass (Aegilops cylindrica Host.), we carried out mixed pollinations in experimental and natural conditions. A set of species-specific RAPD (random amplified polymorphic DNA) and microsatellite markers were used to detect the presence of parental markers in the progeny of the plants used in these experiments. No hybrids were found within the offsprings of the plants used for the greenhouse experiments, while 85 Ae. cylindrica×T. æstivum hybrids were found within 2400 analyzed F₁ plants resulting from the field pollinations. The hybridization rates for individuals of different populations of the wild species differed considerably: 1% for two populations known for more than 90 years versus 7% for a newly discovered population. Most of the hybrids were completely sterile, but five of them produced 13 seeds (BC₁) by backcross with Ae. cylindrica. Twelve seeds germinated and generated viable and partly fertile plants. About 25% of the wheat specific RAPD markers were found in the BC₁ plants, indicating that introgression of wheat DNA into Ae. cylindrica is possible. In addition, one microsatellite marker, known to be situated on the D genome (a genome shared by both species), was also found in the BC₁ plants.     

Genetic characterization of hybridization and introgression between anadromous rainbow trout (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki)

Interspecific hybridization represents a dynamic evolutionary phenomenon and major conservation problem in salmonid fishes. In this study we used amplified fragment length polymorphisms (AFLP) and mitochondrial DNA (mtDNA) markers to describe the extent and characterize the pattern of hybridization and introgression between coastal rainbow trout (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki). Hybrid individuals were initially identified using principle coordinate analysis of 133 polymorphic AFLP markers. Subsequent analysis using 23 diagnostic AFLP markers revealed the presence of F1, rainbow trout backcross, cutthroat trout backcross and later-generation hybrids. mtDNA analysis demonstrated equal numbers of F1 hybrids with rainbow and cutthroat trout mtDNA indicating reciprocal mating of the parental types. In contrast, rainbow and cutthroat trout backcross hybrids always exhibited the mtDNA from the recurrent parent, indicating a male hybrid mating with a pure female. This study illustrates the usefulness of the AFLP technique for generating large numbers of species diagnostic markers. The pattern of hybridization raises many questions concerning the existence and action of reproductive isolating mechanisms between these two species. Our findings are consistent with the hypothesis that introgression between anadromous populations of coastal rainbow and coastal cutthroat trout is limited by an environment-dependent reduction in hybrid fitness.     

Genetic basis of hybrid male sterility among three closely related species of Drosophila

The genetic basis of hybrid male sterility among three closely related species, Drosophila bipectinata, D. parabipectinata and D. malerkotliana has been investigated by using backcross analysis methods. The role of Y chromosome, major hybrid sterility (MHS) genes (genetic factors) and cytoplasm (non-genetic factor) have been studied in the hybrids of these three species. In the species pair, bipectinata--parabipectinata, Y chromosome introgression of parabipectinata in the genomic background of bipectinata and the reciprocal Y chromosome introgression were unsuccessful as all males in second backcross generation were sterile. Neither MHS genes nor cytoplasm was found important for sterility. This suggests the involvement of X-Y, X-autosomes or polygenic interactions in hybrid male sterility. In bipectinata--malerkotliana and parabipectinata--malerkotliana species pairs, Y chromosome substitution in reciprocal crosses did not affect male fertility. Backcross analyses also show no involvement of MHS genes or cytoplasm in hybrid male sterility in these two species pairs. Therefore, X- autosome interaction or polygenic interaction is supposed to be involved in hybrid male sterility in these two species pairs. These findings also provide evidence that even in closely related species, genetic interactions underlying hybrid male sterility may vary.     

Do escaped transgenes persist in nature? The case of an herbicide resistance transgene in a weedy Brassica rapa population

The existence of transgenic hybrids resulting from transgene escape from genetically modified (GM) crops to wild or weedy relatives is well documented but the fate of the transgene over time in recipient wild species populations is still relatively unknown. This is the first report of the persistence and apparent introgression, i.e. stable incorporation of genes from one differentiated gene pool into another, of an herbicide resistance transgene from Brassica napus into the gene pool of its weedy relative, Brassica rapa , monitored under natural commercial field conditions. Hybridization between glyphosate-resistant [herbicide resistance (HR)] B. napus and B. rapa was first observed at two Québec sites, Ste Agathe and St Henri, in 2001. B. rapa populations at these two locations were monitored in 2002, 2003 and 2005 for the presence of hybrids and transgene persistence. Hybrid numbers decreased over the 3-year period, from 85 out of ~200 plants surveyed in 2002 to only five out of 200 plants in 2005 (St Henri site). Most hybrids had the HR trait, reduced male fertility, intermediate genome structure, and presence of both species-specific amplified fragment length polymorphism markers. Both F₁ and backcross hybrid generations were detected. One introgressed individual, i.e. with the HR trait and diploid ploidy level of B. rapa, was observed in 2005. The latter had reduced pollen viability but produced ~480 seeds. Forty-eight of the 50 progeny grown from this plant were diploid with high pollen viability and 22 had the transgene (1:1 segregation). These observations confirm the persistence of the HR trait over time. Persistence occurred over a 6-year period, in the absence of herbicide selection pressure (with the exception of possible exposure to glyphosate in 2002), and in spite of the fitness cost associated with hybridization.     

A cytonuclear incompatibility causes anther sterility in Mimulus hybrids

Multilocus interactions (also known as Dobzhansky-Muller incompatibilities) are thought to be the major source of hybrid inviability and sterility. Because cytoplasmic and nuclear genomes have conflicting evolutionary interests and are often highly coevolved, cytonuclear incompatibilities may be among the first to develop in incipient species. Here, we report the discovery of cytoplasm-dependent anther sterility in hybrids between closely related Mimulus species, outcrossing M. guttatus and selfing M. nasutus. A novel pollenless anther phenotype was observed in F2 hybrids with the M. guttatus cytoplasm (F2G) but not in the reciprocal F2N hybrids, F1 hybrids or parental genotypes. The pattern of phenotypic segregation in the F2G hybrids and two backcross populations fit a Mendelian single-locus recessive model, allowing us to map the underlying nuclear locus to a small region on LG7 of the Mimulus linkage map. Anther sterility was associated with a 20% reduction in flower size in backcross hybrids and we mapped a major cytoplasm-dependent corolla width QTL with its peak at the anther sterility locus. We argue that the cytonuclear anther sterility seen in hybrids reflects the presence of a cryptic cytoplasmic male sterility (CMS) and restorer system within the hermaphroditic M. guttatus population and therefore name the anther sterility locus restorer-of-male-fertility (RMF). The genetic mapping of RMF is a first step toward testing hypotheses about the molecular basis, individual fitness consequences, and ecological context of CMS and restoration in a system without stable CMS-restorer polymorphism (i.e., gynodioecy). The discovery of cryptic CMS in a hermaphroditic wildflower further suggests that selfish cytoplasmic evolution may play an important, but often undetected, role in shaping patterns of hybrid incompatibility and interspecific introgression in plants.