Inbreeding and the genetic variance in floral traits of Mimulus guttatus

The additive genetic variance, V(A), is frequently used as a measure of evolutionary potential in natural plant populations. Many plants inbreed to some extent; a notable observation given that random mating is essential to the model that predicts evolutionary change from V(A). With inbreeding, V(A) is not the only relevant component of genetic variation. Several nonadditive components emerge from the combined effects of inbreeding and genetic dominance. An important empirical question is whether these components are quantitatively significant. We use maximum likelihood estimation to extract estimates for V(A) and the nonadditive 'inbreeding components' from an experimental study of the wildflower Mimulus guttatus. The inbreeding components contribute significantly to four of five floral traits, including several measures of flower size and stigma-anther separation. These results indicate that inbreeding will substantially alter the evolutionary response to natural selection on floral characters.     

Inbreeding depression in an asexual population of Mimulus guttatus

The reproductive mechanism, that is whether an organism outcrosses, selfs or asexually reproduces, has a substantial impact on the amount and pattern of genetic variation. In this study, we estimate genetic variation and genetic load for a predominately asexual population of Mimulus guttatus, and then compare our results to other studies of predominately sexually reproducing (outcrossing and selfing) populations of M. guttatus. The asexual population had low levels of heterozygosity (H_e = 0.03) and low (but significantly non‐zero) inbreeding load, especially when compared with other M. guttatus populations. This differs greatly from the sexual populations of Mimulus that display substantial inbreeding depression. We discuss a variety of reasons why we see such low load in this study and suggest future research projects to further explore the questions.     

Inbreeding in Mimulus guttatus Reduces Visitation by Bumble Bee Pollinators

Inbreeding in plants typically reduces individual fitness but may also alter ecological interactions. This study examined the effect of inbreeding in the mixed-mating annual Mimulus guttatus on visitation by pollinators (Bombus impatiens) in greenhouse experiments. Previous studies of M. guttatus have shown that inbreeding reduced corolla size, flower number, and pollen quantity and quality. Using controlled crosses, we produced inbred and outbred families from three different M. guttatus populations. We recorded the plant genotypes that bees visited and the number of flowers probed per visit. In our first experiment, bees were 31% more likely to visit outbred plants than those selfed for one generation and 43% more likely to visit outbred plants than those selfed for two generations. Inbreeding had only a small effect on the number of flowers probed once bees arrived at a genotype. These differences were explained partially by differences in mean floral display and mean flower size, but even when these variables were controlled statistically, the effect of inbreeding remained large and significant. In a second experiment we quantified pollen viability from inbred and self plants. Bees were 37–54% more likely to visit outbred plants, depending on the population, even when controlling for floral display size. Pollen viability proved to be as important as floral display in predicting pollinator visitation in one population, but the overall explanatory power of a multiple regression model was weak. Our data suggested that bees use cues in addition to display size, flower size, and pollen reward quality in their discrimination of inbred plants. Discrimination against inbred plants could have effects on plant fitness and thereby reinforce selection for outcrossing. Inbreeding in plant populations could also reduce resource quality for pollinators, potentially resulting in negative effects on pollinator populations.     

Spatial heterogeneity in the strength of selection against deleterious alleles and the mutation load

According to current estimates of genomic deleterious mutation rates (which are often of the order 0.1-1) the mutation load (defined as a reduction in the average fitness of a population due to the presence of deleterious alleles) may be important in many populations. In this paper, I use multilocus simulations to explore the effect of spatial heterogeneity in the strength of selection against deleterious alleles on the mutation load (for example, it has been suggested that stressful environments may increase the strength of selection). These simulations show contrasted results: in some situations, spatial heterogeneity may greatly reduce the mutation load, due to the fact that migrants coming from demes under stronger selection carry relatively few deleterious alleles, and benefit from a strong advantage within demes under weaker selection (where individuals carry many more deleterious alleles); in other situations, however, deleterious alleles accumulate within demes under stronger selection, due to migration pressure from demes under weaker selection, leading to fitness erosion within those demes. This second situation is more frequent when the productivity of the different demes is proportional to their mean fitness. The effect of spatial heterogeneity is greatly reduced, however, when the response to environmental differences is inconsistent across loci.     

Low impact of germline transposition on the rate of mildly deleterious mutation in Caenorhabditis elegans

Little is known about the role of transposable element (TE) insertion in the production of mutations with mild effects on fitness, the class of mutations thought to be central to the evolution of many basic features of natural populations. We propagated mutation-accumulation (MA) lines of two RNAi-deficient strains of Caenorhabditis elegans that exhibit germline transposition. We show here that the impact of TE activity was to raise the level of mildly deleterious mutation by 2- to 8.5-fold, as estimated from fecundity, longevity, and body length measurements, compared to that observed in a parallel MA experiment with a control strain characterized by a lack of germline transposition. Despite this increase, the rate of mildly deleterious mutation was between one and two orders of magnitude lower than the rate of TE accumulation, which was approximately two new insertions per genome per generation. This study suggests that high rates of TE activity do not necessarily translate into high rates of detectable nonlethal mutation.     

Inbreeding alters resistance to insect herbivory and host plant quality in Mimulus guttatus (Scrophulariaceae)

Previous studies have demonstrated genetic variation for resistance to insect herbivores and host plant quality. The effect of plant mating system, an important determinant of the distribution of genetic variation, on host plant characteristics has received almost no attention. This study used a controlled greenhouse experiment to examine the effect of self- and cross-pollination in Mimulus guttatus (Scrophulariaceae) on resistance to and host plant quality for the xylem-feeding spittlebug Philaenus spumarius (Homoptera: Cercopidae). Spittlebugs were found to have a negative effect on two important fitness components in M. guttatus, flower production and above ground biomass. One of two M. guttatus populations examined showed a significant interaction between the pollination and herbivore treatments. In this case, the detrimental effects of herbivores on biomass and flower production were much more pronounced in inbred (self) plants. The presence of spittlebug nymphs increased inbreeding depression by as much as three times. Pollination treatments also had significant effects on important components of herbivore fitness, but these effects were in opposite directions in our two host plant populations. Spittlebug nymphs maturing on self plants emerged as significantly larger adults in one of our host plant populations, indicating that inbreeding increased host plant quality. In our second host plant population, spittlebug nymphs took significantly longer to develop to adulthood on self plants, indicating that inbreeding decreased host plant quality. Taken together these results suggest that the degree of inbreeding in host plant populations can have important and perhaps complex effects on the dynamics of plant-herbivore interactions and on mating-system evolution in the host.     

On the average coefficient of dominance of deleterious spontaneous mutations

T. Mukai and co-workers in the late 1960s and O. Ohnishi in the 1970s carried out a series of experiments to obtain direct estimates of the average coefficient of dominance (h) of minor viability mutations in Drosophila melanogaster. The results of these experiments, although inconsistent, have been interpreted as indicating slight recessivity of deleterious mutations, with h approximately 0.4. Mukai obtained conflicting results depending on the type of heterozygotes used, some estimates suggesting overdominance and others partial dominance. Ohnishi's estimates, based on the ratio of heterozygous to homozygous viability declines, were more consistent, pointing to the above value. However, we have reanalyzed Ohnishi's data, estimating h by the regression method, and obtained a much smaller estimate of approximately 0.1. This significant difference can be due partly to the different weighting implicit in the estimates, but we suggest that this is not the only explanation. We propose as a plausible hypothesis that a putative nonmutational decline in viability occurring in the first half of Ohnishi's experiment (affecting both homozygotes and heterozygotes) has biased upward the estimates from the ratio, while it would not bias the regression estimates. This hypothesis also explains the very high h approximately 0.7 observed in Ohnishi's high-viability chromosomes. By constructing a model of spontaneous mutations using parameters in the literature, we investigate the above possibility. The results indicate that a model of few mutations with moderately large effects and h approximately 0.2 is able to explain the observed estimates and the distributions of homozygous and heterozygous viabilities. Accounting for an expression of mutations in genotypes with the balancer chromosome Cy does not alter the conclusions qualitatively.     

Inbreeding depression due to mildly deleterious mutations in finite populations: size does matter

We studied the effects of population size on the inbreeding depression and genetic load caused by deleterious mutations at a single locus. Analysis shows how the inbreeding depression decreases as population size becomes smaller and/or the rate of inbreeding increases. This pattern contrasts with that for the load, which increases as population size becomes smaller but decreases as inbreeding rate goes up. The depression and load both approach asymptotic limits when the population size becomes very large or very small. Numerical results show that the transition between the small and the large population regimes is quite rapid, and occurs largely over a range of population sizes that vary by a factor of 10. The effects of drift on inbreeding depression may bias some estimates of the genomic rate of deleterious mutation. These effects could also be important in the evolution of breeding systems in hermaphroditic organisms and in the conservation of endangered populations.     

Natural variation for drought-response traits in the Mimulus guttatus species complex

Soil moisture is a key factor affecting plant abundance and distribution, both across and within species. In response to water limitation, plants have evolved numerous morphological, physiological, and phenological adaptations. In both well-watered and water-limited conditions, we identified considerable natural variation in drought-related whole-plant and leaf-level traits among closely related members of the Mimulus guttatus species complex that occupy a diversity of habitats in the field. The self-fertilizing Mimulus nasutus and serpentine-endemic Mimulus nudatus demonstrated the overall greatest tolerance to soil water limitation, exhibiting the smallest reduction in seed set relative to well-watered conditions. This may be due in part to early flowering, faster fruit development, and low stomatal density. In contrast, flowering of coastal M. guttatus was so delayed that it precluded any seed production in water-limited conditions. This range of phenotypic responses to soil water deficit in Mimulus, coupled with developing genomic resources, holds considerable promise for identifying genomic variation responsible for adaptive responses to soil water availability.     

Analysis of the estimators of the average coefficient of dominance of deleterious mutations

We investigate the sources of bias that affect the most commonly used methods of estimation of the average degree of dominance (h) of deleterious mutations, focusing on estimates from segregating populations. The main emphasis is on the effect of the finite size of the populations, but other sources of bias are also considered. Using diffusion approximations to the distribution of gene frequencies in finite populations as well as stochastic simulations, we assess the behavior of the estimators obtained from populations at mutation-selection-drift balance under different mutational scenarios and compare averages of h for newly arisen and segregating mutations. Because of genetic drift, the inferences concerning newly arisen mutations based on the mutation-selection balance theory can have substantial upward bias depending upon the distribution of h. In addition, estimates usually refer to h weighted by the homozygous deleterious effect in different ways, so that inferences are complicated when these two variables are negatively correlated. Due to both sources of bias, the widely used regression of heterozygous on homozygous means underestimates the arithmetic mean of h for segregating mutations, in contrast to their repeatedly assumed equality in the literature. We conclude that none of the estimators from segregating populations provides, under general conditions, a useful tool to ascertain the properties of the degree of dominance, either for segregating or for newly arisen deleterious mutations. Direct estimates of the average h from mutation-accumulation experiments are shown to suffer some bias caused by purging selection but, because they do not require assumptions on the causes maintaining segregating variation, they appear to give a more reliable average dominance for newly arisen mutations.     

Isolation of a copper complex and its rate of appearance in roots of Mimulus guttatus

A Cu-complex was isolated from the roots of copper-tolerant Mimulus guttatus. The elution volume of the complex determined by gel permeation chromatography was similar to that of rat-liver cadmium thionein. The complex was heat stable, had a relatively high ratio of absorbance at 254 nm: 280 nm and incorporated ³⁵S. The complex, purified using a combination of gel permeation chromatography and anion-exchange chromatography, contained more glutamine/glutamic acid and glycine residues than mammalian metallothioneins. The amount of the complex in roots increased after 5 h growth in a solution containing 16 M Cu. Induction was preceded by an increase in the concentrations in root tissue of unknown compounds containing sulphur which may serve as precursors. The availability of these compounds appeared to regulate the rate of synthesis of this Cu-complex.Abbreviations CuBP copper-binding protein - HPLC highperformance liquid chromatography - MT metallothionein - Th thionein - Tris 2-amino-2-(hydroxymethyl)1-3-propanediol     

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.     

Testing for increased mutation rate for neutral alleles

Methods are given to test whether, at a selectively neutral locus, one can reasonably assume a recent increase in mutation rate to new alleles. The tests use the number of singletons, doubletons, etc. occurring at this locus and also the frequency of the most frequent allele.     

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.     

Inbreeding rate modifies the dynamics of genetic load in small populations

The negative fitness consequences of close inbreeding are widely recognized, but predicting the long-term effects of inbreeding and genetic drift due to limited population size is not straightforward. As the frequency and homozygosity of recessive deleterious alleles increase, selection can remove (purge) them from a population, reducing the genetic load. At the same time, small population size relaxes selection against mildly harmful mutations, which may lead to accumulation of genetic load. The efficiency of purging and the accumulation of mutations both depend on the rate of inbreeding (i.e., population size) and on the nature of mutations. We studied how increasing levels of inbreeding affect offspring production and extinction in experimental Drosophila littoralis populations replicated in two sizes, N = 10 and N = 40. Offspring production and extinction were measured over 25 generations concurrently with a large control population. In the N = 10 populations, offspring production decreased strongly at low levels of inbreeding, then recovered only to show a consistent subsequent decline, suggesting early expression and purging of recessive highly deleterious alleles and subsequent accumulation of mildly harmful mutations. In the N = 40 populations, offspring production declined only after inbreeding reached higher levels, suggesting that inbreeding and genetic drift pose a smaller threat to population fitness when inbreeding is slow. Our results suggest that highly deleterious alleles can be purged in small populations already at low levels of inbreeding, but that purging does not protect the small populations from eventual genetic deterioration and extinction.     

Pollen limitation and natural selection on floral characters in the yellow monkeyflower, Mimulus guttatus

In flowering plants, pollen limitation has been proposed to intensify selection on floral characters important in pollinator attraction, but may also select for traits that increase seed set through autonomous selfing. Here, a factorial design (+/- pollen addition, +/- pollinator removal) was used to investigate how the pollination environment affects selection on floral morphology via female fitness in a mixed-mating population of the yellow monkeyflower, Mimulus guttatus (Phrymaceae). Female fitness was strongly pollen-limited, with supplementally pollinated plants setting 37% more seeds than open-pollinated individuals. Strong positive selection was found on flower length, weak positive selection on flower width : length ratio and no selection on stigma-anther distance in both open-pollinated and supplementally pollinated treatments. By contrast, flowers with relatively narrow corollas and low stigma-anther distances were favored in the pollinator exclusion treatment. These results provide mixed support for the idea that pollen limitation intensifies selection on floral characters. Despite strong phenotypic selection, natural pollen limitation did not mediate selection on characters associated with either pollinator attraction or self-fertilization. However, the novel pattern of selection on severely pollen-limited plants suggests that reproductive assurance against pollinator loss may have been directly involved in the floral evolution of closely related selfing taxa.     

The potential for adaptive evolution of pollen grain size in Mimulus guttatus

We tested whether pollen grain size (PGS) shows heritable variation in three independent populations of Mimulus guttatus by imposing artificial selection for this character. In addition, we looked for correlated responses to selection in a range of 15 other floral characters. Heritable variation in PGS was found in all three populations, with heritabilities of between 19 and 40% (average 30%). After three generations, upward and downward lines differed on average by 30% in pollen volume. No consistent patterns of correlated response were found in other characters, indicating that PGS can respond to selective forces acting on PGS alone. Possible selection mechanisms on PGS in this species could include intermale selection, if large pollen grains produce more competitive gametophytes; or optimization of patterns of resource allocation, if local mate competition varies.