The genetic architecture of interspecific variation in mimulus

The genetic architecture of various floral and morphological differences between Mimulus cupriphilus and Mimulus guttatus is investigated. M. cupriphilus is believed to have speciated from M. guttatus in the recent past. The two parent species, the F(1) and F(2), and two backcrosses were grown and scored for 23 different characters. The analysis of means revealed significant epistasis for a number of the floral characters, particularly those involving the length of parts. Dominance was generally toward M. guttatus, except for the characters related to flowering time. Analysis of the genetic correlations between characters revealed that there were at least four different polygenic genetic systems, governing flowering time, size of flower, number of spots on the corolla, and general size. An analysis of minimum gene number suggested that there were at least 3-7 genes controlling floral size, and a different three controlling floral spot number. Two other characters, corolla lobe shape and stem color, were produced by independent major gene differences. Annuality was also shown to be heritable. The two species appear to utilize the same gene for copper tolerance. The results are discussed in the light of current theories of speciation.     

Factors affecting the co-existence of the serpentine endemic Mimulus nudatus Curran and its presumed progenitor, Mimulus guttatus Fischer ex DC

Population genetics theory suggests that two species which are reproductively isolated only by postzygotic barriers cannot co-exist in sympatry since the rarer population will become extinct. Mimulus nudatus and Mimulus guttatus are two closely related species that are isolated by a postzygotic barrier operating at the seed provisioning stage. On several sites on die serpentine soils of Lake and Napa counties, California, M. nudatus and M. guttatus live in sympatry and flowering times of the species overlap, so the species are sometimes flowering next to each other. We investigated whether there was any reduction in fertility of M. nudatus and M. guttatus caused by interspecific crosses when growing in sympatry. The pollinators of M. nudatus and M. guttatus were identified. Small sweat bees, Dialictus sp., preferentially visited the smaller flowered M. nudatus species and honey bees, Apis mellifera , preferred the larger flowered M. guttatus . In spite of most pollinator visits being intra-specinc, individuals of bodi bee species made transitions between the Mimulus species. This will result in greater pollen transfer from M. guttatus to M. nudatus than vice versa because firstly, M. guttatus produces more pollen and secondly, the sweat bees were too small to touch the stigma of a M. guttatus flower. This asymmetry in gene flow was detected by a greater reduction in viable seed produced by M. nudatus plants when surrounded by M. guttatus plants dian vice versa. Only when M. nudatus was the maternal parent could any hybrids be detected in field produced seed. To enable M. nudatus and M. guttatus to co-exist in sympatry, the two species may thus need to be sufficiendy ecologically different. Such ecological differentiation was attributed to the greater drought tolerance of M. nudatus . In addition, greenhouse experiments suggested that M. nudatus may have evolved greater tolerance to calcium deficient soils.     

Nickel tolerance and copper - nickel co - tolerance in Mimulus guttatus from copper mine and serpentine habitats

Previous work on M. guttatus suggested that nickel tolerance in copper mine populations may also be given by the genes for copper tolerance. It has been shown that copper tolerance in M. guttatus is controlled by a single major gene, plus a number of minor genes (or modifiers) which elevate copper tolerance. Crosses between nickel tolerant individuals from three families and non - tolerants showed that nickel tolerance in M. guttatus is heritable. In order to study the effects of the major copper tolerance gene on copper - nickel co - tolerance in M. guttatus, homozygous copper tolerant and non - tolerant lines were screened against nickel. Significant differences occur between these lines for copper, but were not found when analysed for nickel, indicating that copper - nickel tolerance is not governed by the major gene for copper tolerance. To test whether the minor genes for copper have a pleiotropic effect on nickel tolerance, five selection lines derived from three copper mines (Copperopolis, Penn and Quail) in Calaveras county, California, which vary in degree of tolerance to copper, by the presence or absence of minor copper genes, were also screened against nickel. Two out of three of the lines from Copperopolis showed elevated tolerance to nickel, but two further selection lines derived from Penn and Quail copper mines gave no indication of increased nickel tolerance. These results suggest that the minor genes for copper do not give tolerance to nickel. This was confirmed by the screening of modifier lines, in which modifiers for differing degrees of copper tolerance were inserted into a non - tolerant background. Genotypes possessing fewer copper modifiers yielded higher nickel tolerance than those genotypes which have a greater number of modifiers. Thus nickel tolerance in this species is heritable and under the control of different genes to those producing copper tolerance.     

Pollen tube competition as a mechanism of prezygotic reproductive isolation between Mimulus nasutus and its presumed progenitor M. guttatus

This paper considers the extent to which differences in pollen tube growth rates can provide prezygotic reproductive isolation between Mimulus nasutus and its presumed progenitor, Mimulus guttatus . Mimulus nasutus is partially cleistogamous, but its larger chasmogamous flowers offer appreciable opportunity for outcrossing. Mimulus nasutus was found to have smaller pollen grains and shorter styles than M. guttatus . No differences were observed in pollen grain germination on conspecific and heterospecific stigmas. However, pollen tube growth rates of M. nasutus were found to be much slower than those of M. guttatus in the styles of that species. Consequently, any M. nasutus pollen transferred to an M. guttatus stigma was found to be competitively disadvantaged in an M. gutattus style. By contrast, no difference in pollen tube growth rate was detected between the species when growing in M. nasutus styles, possibly because M. nasutus styles are unable to support fast pollen tube growth. We tested the prediction from the pollen tube studies that a 50:50 mix of M. guttatus and M. nasutus pollen would produce 50% hybrid seeds when M. nasutus was the maternal parent, and near to 0% hybrid seed when M. guttatus was the maternal parent. The results were found to support this prediction. We conclude that pollen–pistil interactions can effect strong reproductive isolation between these species, as M. guttatus pollen tubes have a competitive advantage over those of M. nasutus in an M. guttatus style, but not in an M. nasutus style.     

Can an increased copper requirement in copper-tolerant Mimulus guttatus explain the cost of tolerance? II. Reproductive phase

Circumstantial evidence suggests that plants that have evolved metal tolerance are at a disadvantage on normal soil, i.e. there is a cost of tolerance. One hypothesis for the cause of this cost is that individuals have a greater requirement for copper, and so suffer micronutrient deficiency on normal soils, as a result of a reduced uptake, distribution and/or utilization of copper. We provided highly and less copper-tolerant plants of Mimulus guttatus Fischer ex DC. (the common monkey flower) with sub-optimal copper, and demonstrated the importance of copper as an essential micronutrient during the reproductive phase, both in the production of viable pollen and in seed set. We also looked at the effect of sub-optimal copper supply on the growth of the microgametophyte, and the efficiency with which seed was set. No evidence was found that highly tolerant plants have an increased copper requirement during the reproductive phase. This is in agreement with earlier work on Mimulus guttatus , which investigated the copper requirement of highly tolerant plants during vegetative growth and found that any differences in copper requirement were small. The 'metal requirement hypothesis' is, therefore, not the sole explanation for the cost of copper tolerance in M. guttatus .     

Developmental analysis of the evolutionary origin of vegetative propagules in Mimulus gemmiparus (Scrophulariaceae)

Mimulus gemmiparus (Scrophulariaceae), a rare endemic of Colorado, has a novel life history that depends on an unusual method of vegetative reproduction. The plants are functionally annuals; however, reproduction is asexual via propagules that have been termed gemmae. The morphological identity and the evolutionary antecedent of these propagules are unclear. We approached this problem through comparative developmental analyses of M. gemmiparus and the presumed progenitor species, Mimulus guttatus. In M. gemmiparus there are two meristems initiated in the axil of each leaf primordium. The distal meristem has the potential to produce either a lateral branch or a flower, and the proximal meristem becomes a vegetative propagule (the gemma) that is ultimately surrounded by an expanded, ensheathing petiole. The first leaves of the propagules are thickened and are the site of nutrient storage. Consequently, these propagules can be characterized morphologically as brood bulbils. Mimulus guttatus also has two meristems in each leaf axil; however, the proximal meristem typically remains dormant and serves no function in the life history of this species. Based on architectural and developmental correspondence, we hypothesize that the propagule of M. gemmiparus is homologous to the proximal meristem of M. guttatus. Comparative analysis shows that evolution of the bulbil has involved both the incorporation of features present in shoots of M. guttatus and the acquisition of novel features.     

Toward the evolutionary genomics of gametophytic divergence: patterns of transmission ratio distortion in monkeyflower (Mimulus) hybrids reveal a complex genetic basis for conspecific pollen precedence

Conspecific pollen precedence (CPP) is a major component of reproductive isolation between many flowering plant taxa and may reveal mechanisms of gametophytic evolution within species, but little is known about the genetic basis and evolutionary history of CPP. We systematically investigated the genetic architecture of CPP using patterns of transmission ratio distortion (TRD) in F2 and backcross hybrids between closely related species of Mimulus (Phrymaceae) with divergent mating systems. We found that CPP in Mimulus hybrids was polygenic and was the majority source of interspecific TRD genome-wide, with at least eight genomic regions contributing to the transmission advantage of M. guttatus pollen grains on M. guttatus styles. In aggregate, these male-specific transmission ratio distorting loci (TRDLs) were more than sufficient to account for the 100% precedence of pure M. guttatus pollen over M. nasutus pollen in mixed pollinations of M. guttatus. All but one of these pollen TRDLs were style-dependent; that is, we observed pollen TRD in F(1) and/or M. guttatus styles, but not in M. nasutus styles. These findings suggest that species-specific differences in pollen tube performance accumulate gradually and may have been driven by coevolution between pollen and style in the predominantly outcrossing M. guttatus.     

Patterns of nucleotide variation and reproductive isolation between a Mimulus allotetraploid and its progenitor species

Here we report our characterization of a widespread, highly selfing Mimulus allotetraploid formed by interspecific hybridization between M. nasutus and M. guttatus. Nucleotide variation at two nuclear loci (mCYCA and mAP3) within and among tetraploid populations resolves two haplotype clusters for each locus: one shares near identity with sequences from M. nasutus and the other group shares substantial variation with M. guttatus. With respect to the two loci studied, each allotetraploid individual is a 'fixed heterozygote' carrying sequences from both clusters. Moreover, mCYCA variation is consistent with at least two evolutionary origins for the Mimulus allotetraploid. We show that the allotetraploid is strongly reproductively isolated from M. nasutus and M. guttatus; interploidy crosses produce almost no viable seeds. By extension, we infer strong triploid block and argue that Mimulus allotetraploid formation might proceed in one step via the union of unreduced gametes in an M. nasutus-M. guttatus F(1) hybrid. We also discuss the potential roles of mating system and flowering asynchrony in allotetraploid establishment.     

Patterns of nucleotide diversity in two species of Mimulus are affected by mating system and asymmetric introgression

The evolutionary transition from outcrossing to self-fertilization has far-reaching implications for patterns of intraspecific genetic diversity and the potential for speciation. Using DNA sequence variation at two nuclear loci, we examined the divergence history of two closely related species of Mimulus. To investigate the effects of mating system and introgressive hybridization on the outcrossing M. guttatus and the selfing M. nasutus, we inspected nucleotide diversity within and between natural populations spanning the species' geographic ranges. High sequence similarity among populations of the selfing M. nasutus points to a single evolutionary origin for the species. Consistent with their distinct mating systems, all genetic variation in M. nasutus is distributed among populations, whereas M. guttatus exhibits appreciable levels of nucleotide diversity within populations. Silent genetic diversity is extensive in M. guttatus (mean theta(sil)/site = 0.077) and greatly exceeds the predicted twofold elevation in neutral variation for outcrossers relative to selfers. The finding of several M. guttatus sequences that share complete identity with sequences from M. nasutus suggests that recent asymmetric introgression may have occurred. We argue that exceptionally high nucleotide diversity in M. guttatus is consistent with a long-term history of directional introgression from M. nasutus to M. guttatus throughout the divergence of these two species.     

Natural variation for a hybrid incompatibility between two species of Mimulus

Understanding the process by which hybrid incompatibility alleles become established in natural populations remains a major challenge to evolutionary biology. Previously, we discovered a two-locus Dobzhansky-Muller incompatibility that causes severe hybrid male sterility between two inbred lines of the incompletely isolated wildflower species, Mimulus guttatus and M. nasutus. An interspecific cross between these two inbred lines revealed that the M. guttatus (IM62) allele at hybrid male sterility 1 (hms1) acts dominantly in combination with recessive M. nasutus (SF5) alleles at hybrid male sterility 2 (hms2) to cause nearly complete hybrid male sterility. In this report, we extend these genetic analyses to investigate intraspecific variation for the hms1-hms2 incompatibility in natural populations of M. nasutus and M. guttatus, performing a series of interspecific crosses between individuals collected from a variety of geographic locales. Our results suggest that hms2 incompatibility alleles are common and geographically widespread within M. nasutus, but absent or rare in M. guttatus. In contrast, the hms1 locus is polymorphic within M. guttatus and the incompatibility allele appears to be extremely geographically restricted. We found evidence for the presence of the hms1 incompatibility allele in only two M. guttatus populations that exist within a few kilometers of each other. The restricted distribution of the hms1 incompatibility allele might currently limit the potential for the hms1-hms2 incompatibility to act as a species barrier between sympatric populations of M. guttatus and M. nasutus. Extensive sampling within a single M. guttatus population revealed that the hms1 locus is polymorphic and that the incompatibility allele appears to segregate at intermediate frequency, a pattern that is consistent with either genetic drift or natural selection.     

Transmission ratio distortion in intraspecific hybrids of Mimulus guttatus: implications for genomic divergence

We constructed a genetic linkage map between two divergent populations of Mimulus guttatus. We genotyped an F(2) mapping population (N = 539) at 154 AFLP, microsatellite, and gene-based markers. A framework map was constructed consisting of 112 marker loci on 14 linkage groups with a total map length of 1518 cM Kosambi. Nearly half of all markers (48%) exhibited significant transmission ratio distortion (alpha = 0.05). By using a Bayesian multipoint mapping method and visual inspection of significantly distorted markers, we detected 12 transmission ratio distorting loci (TRDL) throughout the genome. The high degree of segregation distortion detected in this intraspecific map indicates substantial genomic divergence that perhaps suggests genomic incompatibilities between these two populations. We compare the pattern of transmission ratio distortion in this map to an interspecific map constructed between M. guttatus and M. nasutus. A similar level of segregation distortion is detected in both maps. Collinear regions between maps are compared to determine if there are shared genetic patterns of non-Mendelian segregation distortion within and among Mimulus species.     

Five anthocyanin polymorphisms are associated with an R2R3-MYB cluster in Mimulus guttatus (Phrymaceae)

? Premise of study: Botanists have long been interested in the reasons for genetic variation among individuals, populations, and species of plants. The anthocyanin pathway is ideal for studying the evolution of such phenotypic variation. ? Methods: We used a combination of quantitative trait loci mapping and association studies to understand the genetic basis of variation in five anthocyanin phenotypes including calyx, corolla, and leaf coloration patterns that vary within and among populations of Mimulus guttatus. We then examined what genes might be responsible for this phenotypic variation and whether one of the traits, calyx spotting, is randomly distributed across the geographic range of the species. ? Key results: All five phenotypes in M. guttatus were primarily controlled by the same major locus (PLA1), which contains a tandem array of three R2R3-MYB genes known to be involved in the evolution of flower color in a related species of Mimulus. Calyx spotting was nonrandomly distributed across the range of M. guttatus and correlated with multiple climate variables. ? Conclusions: The results of this study suggest that variation in R2R3-MYB genes is the primary cause of potentially important anthocyanin phenotypic variation within and among populations of M. guttatus, a finding consistent with recent theoretical and empirical research on flower color evolution.     

Polymorphic microsatellite loci in Mimulus guttatus and related species

The evolution of self-fertilization from outcrossing is one of the most common evolutionary transitions in flowering plants. One goal of our research is to develop a linkage map based upon microsatellite loci in Mimulus guttatus and the selfing derivative M. nasutus in order to examine the genetics of one such transition. Here we present 19 primer pairs that detect polymorphisms between these two species. Within-population allelic surveys demonstrate the value of these markers for detailed analysis of population structure. We have found also that many of the microsatellites are conserved in more distantly related Mimulus species, and may thus serve as useful genetic markers for population genetic and comparative mapping studies within this diverse genus.     

A genetic map in the Mimulus guttatus species complex reveals transmission ratio distortion due to heterospecific interactions.

As part of a study of the genetics of floral adaptation and speciation in the Mimulus guttatus species complex, we constructed a genetic linkage map of an interspecific cross between M. guttatus and M. nasutus. We genotyped an F(2) mapping population (N = 526) at 255 AFLP, microsatellite, and gene-based markers and derived a framework map through repeated rounds of ordering and marker elimination. The final framework map consists of 174 marker loci on 14 linkage groups with a total map length of 1780 cM Kosambi. Genome length estimates (2011-2096 cM) indicate that this map provides thorough coverage of the hybrid genome, an important consideration for QTL mapping. Nearly half of the markers in the full data set (49%) and on the framework map (48%) exhibited significant transmission ratio distortion (alpha = 0.05). We localized a minimum of 11 transmission ratio distorting loci (TRDLs) throughout the genome, 9 of which generate an excess of M. guttatus alleles and a deficit of M. nasutus alleles. This pattern indicates that the transmission ratio distortion results from particular interactions between the heterospecific genomes and suggests that substantial genetic divergence has occurred between these Mimulus species. We discuss possible causes of the unequal representation of parental genomes in the F(2) generation.     

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.     

Pleiotropic quantitative trait loci contribute to population divergence in traits associated with life-history variation in Mimulus guttatus

Evolutionary biologists seek to understand the genetic basis for multivariate phenotypic divergence. We constructed an F2 mapping population (N = 539) between two distinct populations of Mimulus guttatus. We measured 20 floral, vegetative, and life-history characters on parents and F1 and F2 hybrids in a common garden experiment. We employed multitrait composite interval mapping to determine the number, effect, and degree of pleiotropy in quantitative trait loci (QTL) affecting divergence in floral, vegetative, and life-history characters. We detected 16 QTL affecting floral traits; 7 affecting vegetative traits; and 5 affecting selected floral, vegetative, and life-history traits. Floral and vegetative traits are clearly polygenic. We detected a few major QTL, with all remaining QTL of small effect. Most detected QTL are pleiotropic, implying that the evolutionary shift between these annual and perennial populations is constrained. We also compared the genetic architecture controlling floral trait divergence both within (our intraspecific study) and between species, on the basis of a previously published analysis of M. guttatus and M. nasutus. Eleven of our 16 floral QTL map to approximately the same location in the interspecific map based on shared, collinear markers, implying that there may be a shared genetic basis for floral divergence within and among species of Mimulus.     

A simple genetic incompatibility causes hybrid male sterility in mimulus

Much evidence has shown that postzygotic reproductive isolation (hybrid inviability or sterility) evolves by the accumulation of interlocus incompatibilities between diverging populations. Although in theory only a single pair of incompatible loci is needed to isolate species, empirical work in Drosophila has revealed that hybrid fertility problems often are highly polygenic and complex. In this article we investigate the genetic basis of hybrid sterility between two closely related species of monkeyflower, Mimulus guttatus and M. nasutus. In striking contrast to Drosophila systems, we demonstrate that nearly complete hybrid male sterility in Mimulus results from a simple genetic incompatibility between a single pair of heterospecific loci. We have genetically mapped this sterility effect: the M. guttatus allele at the hybrid male sterility 1 (hms1) locus acts dominantly in combination with recessive M. nasutus alleles at the hybrid male sterility 2 (hms2) locus to cause nearly complete hybrid male sterility. In a preliminary screen to find additional small-effect male sterility factors, we identified one additional locus that also contributes to some of the variation in hybrid male fertility. Interestingly, hms1 and hms2 also cause a significant reduction in hybrid female fertility, suggesting that sex-specific hybrid defects might share a common genetic basis. This possibility is supported by our discovery that recombination is reduced dramatically in a cross involving a parent with the hms1-hms2 incompatibility.     

Cytoplasmic male sterility in Mimulus hybrids has pleiotropic effects on corolla and pistil traits

The mechanisms underlying genetic associations have important consequences for evolutionary outcomes, but distinguishing linkage from pleiotropy is often difficult. Here, we use a fine mapping approach to determine the genetic basis of association between cytonuclear male sterility and other floral traits in Mimulus hybrids. Previous work has shown that male sterility in hybrids between Mimulus guttatus and Mimulus nasutus is due to interactions between a mitochondrial gene from M. guttatus and two tightly linked nuclear restorer alleles on Linkage Group 7, and that male sterility is associated with reduced corolla size. In the present study, we generated a set of nearly isogenic lines segregating for the restorer region and male sterility, but with unique flanking introgressions. Male-sterile flowers had significantly smaller corollas, longer styles and greater stigmatic exsertion than fertile flowers. Because these effects were significant regardless of the genotypic composition of introgressions flanking the restorer region, they suggest that these floral differences are a direct byproduct of the genetic incompatibility causing anther abortion. In addition, we found a non-significant but intriguing trend for male-sterile plants to produce more seeds per flower than fertile siblings after supplemental pollination. Such pleiotropic effects may underlie the corolla dimorphism frequently observed in gynodioecious taxa and may affect selection on cytoplasmic male sterility genes when they initially arise.     

Heterospecific pollen deposition: does diversity alter the consequences?

? In natural communities, plants can receive pollen from multiple heterospecifics as well as conspecifics. However, studies on the effects of interspecific pollen transfer have focused on interactions between species pairs. The potential exists for diverse interactions among heterospecific pollen (HP) grains on the stigma, and for these to affect plant reproduction, alone or in combination with conspecific pollen (CP) loss, but these interactions have not yet been explored. ? We used hand-pollinations to simulate increasing community diversity and CP loss on Mimulus guttatus stigmas. We used pollen mixes of one to three heterospecific donors to determine how species composition and CP load size affect seed production and to characterize the mechanisms underlying fertilization failure. ? Heterospecific pollen deposition reduced M. guttatus seed production and while the effect increased with the number of heterospecific donors, the strength depended on species composition and was independent of conspecific load size. Different types of interactions (additive and synergistic) are hypothesized to underlie the diverse effects on M. guttatus reproductive success. ? Our results suggest that an increase in the diversity of heterospecific donors will not always lead to a greater decrease in fitness because multispecies effects depend on the interacting species.