COVARIATION OF SELFING RATES WITH PARENTAL GENE FIXATION INDICES WITHIN POPULATIONS OF MIMULUS GUTTATUS

Wright's gene fixation index F and two single-locus effective selfing rates—the selfing rate at loci with fixed alleles, and the selfing rate at loci without fixed alleles—were estimated in five populations of Mimulus guttatus. These two effective selfing rates describe the inbreeding observed at a single locus when both uniparental and biparental inbreeding are practiced. Estimates were made using progeny arrays assayed for six allozyme loci and two morphological loci exhibiting dominance. The average of the two selfing rates computed for subpopulations (ca. 10 m diameter) ranged from 24% to 59%, with a mean of 37%. When computed for populations (ca. 1 km diameter), average selfing rates were about 10% higher. In four populations, the selfing rate at loci with fixed alleles was higher than the selfing rate at loci without fixed alleles. Thus, the covariance of selfing with parental gene fixation was positive. In one of the populations, estimates for individual plants sampled along a transect gave positive correlations for selfing rates and for gene-fixation indices between adjacent plants. A highly positive correlation between selfing rate and gene fixation of individual plants was also observed. In another population, the covariance of selfing with gene fixation was higher for a locus causing leaf spots than for allozyme loci. This covariance is partially caused by 1) variation in homozygosity among neighborhoods and 2) biparental inbreeding within neighborhoods. The consequences of this covariance are discussed.     

EFFECTS OF DIFFERENT LEVELS OF INBREEDING ON FITNESS COMPONENTS IN MIMULUS GUTTATUS

Recent theoretical work has shown that there can be selection favoring the maintenance of sexual reproduction and the evolution of increased recombination when deleterious mutations at different loci interact synergistically, such that the logarithm of fitness declines at a greater than linear rate with the number of harmful mutations per genome. The purpose of this experimental study was to determine whether synergism exists for genes affecting fitness components in two partially selfing populations of the monkey flower Mimulus guttatus. For each wild population, a large randomly mated base population was constructed and many independent lines, inbred to differing degrees, were extracted from this base population. Lines with expected inbreeding coefficients of 0, 0.25, 0.5, and 0.75 were raised simultaneously in the greenhouse and were scored for germination, flowering, flower production, and pollen viability. All fitness traits except germination success declined with increased inbreeding, but in spite of the substantial inbreeding depression found in this study, relatively little evidence of synergistic epistasis was found. The only trait that showed evidence of synergism was pollen viability. These results indicate that synergism is not strong for the fitness components measured in this study. The evidence for synergism from other published studies is also reviewed.     

MULTIPLE PATERNITY AND SELF-FERTILIZATION IN RELATION TO FLORAL AGE IN MIMULUS GUTTATUS (SCROPHULARIACEAE)

To gain some understanding of the mechanisms responsible for the intermediate levels of both multiple paternity and selfing observed previously in the common monkeyflower, Mimulus guttatus, we performed a field experiment to assess the role of 1) multiple paternity through sequential pollinator visits and 2) selfing through corolla abscission. In M. guttatus, flowers remain open for several days; then, in the process of corolla abscission anthers drag past the stigma. We predicted that multiple visitation by pollinators over this period should increase the degree of multiple paternity of sibling arrays, and that corolla dragging may be the primary cause of the observed selfing. In three northern California populations corollas were removed after being open for 1 day (preventing further mating), and the type of matings resulting was compared to matings from adjacent flowers with undisturbed corollas. On average, seed set was twice as great in flowers with unmanipulated corollas, indicating that about half of all matings in the lifespan of a M. guttatus flower occur after 1 day. Pollen supplementation revealed that pollen was limiting in one population. Electrophoretic assay of progeny showed multiple paternity was greater in unmanipulated flowers in the two populations that were not pollen limited, suggesting that sequential pollination may be an important source of multiple paternity. However, no difference was detected in the selfing rate between the two treatments, suggesting that corolla dragging was not a source of selfing even under pollen-limited conditions.     

DEVELOPMENTAL SELECTION IN RESPONSE TO ENVIRONMENTAL CONDITIONS OF THE MATERNAL PARENT IN MIMULUS GUTTATUS

Copper tolerance is expressed in the diploid sporophyte as well as the microgametophyte of Mimulus guttatus. Previous studies, based on reproductive output, suggested that selection for copper tolerance could occur within the pistil. The objective of this study was to determine if selection within the pistil can increase sporophytic tolerance to copper and to determine whether this selection occurs pre- or postzygotically. Mixtures of pollen from copper tolerant or sensitive sources or from plants heterozygous for tolerance to copper were applied at two intensities to plants cloned and grown in control or copper supplemented solutions. The proportion of copper tolerant progeny showed a small, 7%, but significant increase when pollen recipients were grown with added copper. Comparisons of the numbers of tolerant progeny, as well as various components of reproduction, following light and heavy pollinations suggested that microgametophytic selection was unlikely to account for this increase. However, the 8 to 10% decrease in the seed/zygote ratio, compared to control values, was sufficient to account for the difference in proportion of copper tolerant progeny from control and copper treated plants. Thus, it appeared likely that selection for copper tolerance could occur within the pistil, and that much of this selection occurred postzygotically through the early failure of developing seeds.     

THE ROLE OF GENES OF LARGE EFFECT ON INBREEDING DEPRESSION IN MIMULUS GUTTATUS

Severe inbreeding depression is routinely observed in outcrossing species. If inbreeding load is due largely to deleterious alleles of large effect, such as recessive lethals or steriles, then most of it is expected to be purged during brief periods of inbreeding. In contrast, if inbreeding depression is due to the cumulative effects of many deleterious alleles of small effect, then it will be maintained in the face of periodic inbreeding. Whether or not inbreeding depression can be purged with inbreeding in the short term has important implications for the evolution of mating systems and the probability that a small population will go extinct. In this paper I evaluate the extent to which the tremendous inbreeding load in a primarily outcrossing population of the wildflower, Mimulus guttatus, is due to alleles of large effect. To do this, I first constructed a large outbred “ancestral” population by randomly mating plants collected as seeds from a natural population. From this population I formed 1200 lines that were maintained by self-fertilization and single seedling descent: after five generations of selling, 335 lines had survived the inbreeding process. Selection during the line formation is expected to have largely purged alleles of large effect from the collection of highly inbred lines. Because alleles with minor effects on fitness should have been effectively neutral, the inbreeding depression due to this class of genes should have been unchanged. The inbred lines were intercrossed to form a large, outcrossed “purged” population. Finally, I estimated the fitness of outbred and selfed progeny from the ancestral and purged populations to determine the contribution of major deleterious alleles on inbreeding depression. I found that although the average fitness of the outcrossed progeny nearly doubled following purging, the limited decline in inbreeding depression and limited increase in inbred fitness indicates that alleles of large effect are not the principle cause of inbreeding depression in this population. In aggregate, the data suggest that lethals and steriles make a minority contribution to inbreeding depression and that the increased outbred fitness is due primarily to adaptation to greenhouse conditions.     

CORRELATED MATINGS IN THE PARTIAL SELFER MIMULUS GUTTATUS

In partially selfing populations, siblings may be correlated for both selling and paternity. A model of the mating system based upon sampling pairs of progeny from a maternal parent is described. The model separates the correlation of selfing from the correlation of outcrossed paternal alleles and is an approach to paternity analysis suited for larger populations with fewer marker loci. Its parameters determine the components of genetic covariance between sibs and provide information about the average number of fathers in a maternal sibship. Electrophoretic markers were used to obtain estimates of correlated matings for two Mimulus guttatus populations. In both populations, about 50% selfing was observed. For two sibs randomly selected from the same capsule, the correlations of selfing between these sibs were 17% and 12% in the two populations, and the correlations of paternity (the proportion of full-sibs among outcrossed sib-pairs) were 37% and 44%. Sibs from different capsules were not correlated for selfing, and the paternity correlation dropped to near 20% in both populations. However, estimates of correlated matings have high variance, lack statistical independence, and can be difficult to obtain. The use of marker loci with many alleles can alleviate these problems.     

ROLE OF COROLLA ABSCISSION IN DELAYED SELF-POLLINATION OF MIMULUS GUTTATUS (SCROPHULARIACEAE)

Corollas of Mimulus guttatus are shed 1–9 days post-anthesis, and autofertility is often high. The possibility that corolla shedding causes self-pollination in the absence of pollinators was examined. In one experimental population 82% of seed produced under conditions of pollinator exclusion were due to corolla abscission. Corolla abscission, in the absence of pollinators, is a mechanism of delayed self-fertilization in this taxon.     

DELETERIOUS MUTATIONS AND GENETIC VARIATION FOR FLOWER SIZE IN MIMULUS GUTTATUS

Abstract.— Mimulus guttatus is a wildflower that exhibits substantial genetic variation in flower size. Here, we test the hypothesis that this variation is caused by deleterious mutations maintained through mutation‐selection balance. The deleterious‐mutation model predicts that rare, partially recessive alleles will be the primary source of variation. We test this prediction by measuring the change in the mean flower size (ΔM) and the directional dominance of flower size (ΔB) within a selection experiment. If variation is due to rare (partially) recessive alleles, ΔB/ΔM is expected to be positive and exceed one. However, we obtain negative values for ΔB/ΔM from three independent selection lines. This result is statistically inconsistent with the deleterious‐mutation model.     

REPRODUCTIVE ASSURANCE MECHANISMS IN THREE TAXA OF THE MIMULUS GUTTATUS COMPLEX (SCROPHULARIACEAE)

The evolution of inbreeding in plants has often been attributed to selection for the ability to set seed in the absence of mates or pollinators. Mechanisms of reproductive assurance in five populations of mixed mating Mimulus guttatus, three populations of inbreeding M. platycalyx, and two populations of inbreeding M. nasutus were examined in a pollinator-free greenhouse. Reproductive assurance was manifested in all populations by autofertility, vegetative reproduction, or both. The inbreeding taxa had significantly greater levels of autofertility and less vegetative reproduction. Three modes of autofertility were identified: 1) due to corolla abscission only, occurring in three M. guttatus populations; 2) due to both corolla abscission and direct anther-stigma contact by curling of the lower stigmatic lobe into the anthers, occurring in two M. guttatus populations; and 3) direct stigma-anther contact by stigma curling alone prior to corolla abscission, found in each M. platycalyx and M. nasutus population. Stigma-anther distance and its interaction with stigma curling contributed to differences in autofertility among populations. Significant levels of intrapopulation quantitative genetic variation were found for seven of ten traits examined; average levels were similar between inbreeding and mixed mating populations. Genetic variation within populations for autofertility per se was not detected, but significant levels controlling stigma-anther distance were found in two M. guttatus populations. These results show that evolution of inbreeding by natural selection for reproductive assurance is possible in Mimulus, and illustrate the complex changes in floral dynamics and morphology it may involve.     

THE RELATIONSHIP BETWEEN MATING-SYSTEM CHARACTERS AND INBREEDING DEPRESSION IN MIMULUS GUTTATUS

Several recent theoretical considerations of mating-system evolution predict within-population covariation between levels of inbreeding depression and genetically controlled mating-system characters. If inbreeding depression is caused by deleterious recessive alleles, families with characters that promote self-pollination should show lower levels of inbreeding depression than families with characters that promote outcrossing. The converse is expected if inbreeding depression is due to overdominant allelic interactions. Whether these associations between mating-system and viability loci evolve will have important consequences for mating-system evolution. The evolution of selfing within the genus Mimulus is associated with a reduction in stigma-anther separation (i.e., a loss of herkogamy) and high autogamous seed set. In this study we compared families from two M. guttatus populations that differed genetically in their degree of stigma-anther separation. In one of these populations we also compared families that differed genetically in the degree to which they autogamously set seed in a pollinator-free greenhouse. Dams often differed significantly in levels of inbreeding depression for aboveground biomass and flower production, but variation in inbreeding depression was never explained by herkogamy class or autogamy class. Several factors might account for why families with traits associated with selfing did not show lower inbreeding depression, and these are discussed. Our study also demonstrated significant variation among self progeny from a given female likely due to differences in pollination date and position of fruit maturation. The detection of significant dam × sire interactions suggests biparental inbreeding or differences in combining ability for specific pairs of parents.     

DIFFERENTIAL SEED PRODUCTION IN MIMULUS GUTTATUS IN RESPONSE TO INCREASING CONCENTRATIONS OF COPPER IN THE PISTIL BY POLLEN FROM COPPER TOLERANT AND SENSITIVE SOURCES

Selection can occur in the pistil, during a series of stages that include both pre-zygotic (pollen germination, pollen tube growth, and fertilization) and post-zygotic events. This study explores the extent to which selection, at this level, could be due to the environmental conditions under which the maternal parent is growing. Five plants of Mimulus guttatus, tolerant to copper, were vegetatively cloned and each clone was grown in control and in solutions to which copper was added. The maternal plants received pollen from plants either tolerant or sensitive to copper. Seeds and ovules were counted to estimate the number of seeds/capsule, the seed/ovule ratio, the percent fertilization, and the proportion of zygotes aborting for each clone, treatment and pollen source combination. There were large differences among the pollen recipients for each of the measurements. However, there was a consistent pattern to seed production depending on the pollen source and copper treatment. The seed/ovule ratio was unaffected if pollen came from tolerant sources but was reduced by an average of 24% for both copper supplemented treatments if pollen came from copper sensitive sources. Thus, the data indicated that selection due to environmental factors could occur within the pistil. Differences in percent fertilization were not statistically significant, but the seed/zygote ratio showed a pattern that was similar to seed production suggesting that abortion of immature seeds was responsible for most of the difference in seed production.     

EVIDENCE FOR DOBZHANSKY-MULLER INCOMPATIBILITES CONTRIBUTING TO THE STERILITY OF HYBRIDS BETWEEN MIMULUS GUTTATUS AND M. NASUTUS

Abstract Both chromosomal rearrangements and negative interactions among loci (Dobzhansky‐Muller incompatibilities) have been advanced as the genetic mechanism underlying the sterility of interspecific hybrids. These alternatives invoke very different evolutionary histories during speciation and also predict different patterns of sterility in artificial hybrids. Chromosomal rearrangements require drift, inbreeding, or other special conditions for initial fixation and, because heterozygosity per se generates any problems with gamete formation, F1 hybrids will be most infertile. In contrast, Dobzhansky‐Muller incompatibilities may arise as byproducts of adaptive evolution and often affect the segregating F2 generation most severely. To distinguish the effects of these two mechanisms early in divergence, we investigated the quantitative genetics of hybrid sterility in a line cross between two members of the Mimulus guttatus species complex (M. guttatus and M. nasutus). Hybrids showed partial male and female sterility, and the patterns of infertility were not consistent with the action of chromosomal rearrangements alone. F2 and F1 hybrids exhibited equal decreases in pollen viability (> 40%) relative to the highly fertile parental lines. A large excess of completely pollen‐sterile F2 genotypes also pointed to the segregation of Dobzhansky‐Muller incompatibility factors affecting male fertility. Female fertility showed a pattern similarly consistent with epistatic interactions: F2 hybrids produced far fewer seeds per flower than F1 hybrids (88.0 ± 2.8 vs. 162.9 ± 8.5 SE, respectively) and either parental line, and many F2 genotypes were completely female sterile. Dobzhansky‐Muller interactions also resulted in the breakdown of several nonreproductive characters and appear to contribute to correlations between male and female fertility in the F2 generation. These results parallel and contrast with the genetics of postzygotic isolation in model animal systems and are a first step toward understanding the process of speciation in this well‐studied group of flowering plants.     

THE DISTRIBUTION OF POSTMATING REPRODUCTIVE ISOLATING GENES IN POPULATIONS OF THE YELLOW MONKEY FLOWER,MIMULUS GUTTATUS

Postmating reproductive isolating barriers are generally believed to arise as the chance by-product of genetic differentiation. The classical view is that these barriers normally involve differentiation at many loci, and therefore require long periods of allopatric isolation. The formal genetics of, and the distribution of genes responsible for, such barriers are known in very few cases. This paper examines the distribution of the genes responsible for two different postmating barriers in 18 populations of the yellow monkey flower, Mimulus guttatus. The genetic relatedness of the populations was measured by a morphometrical analysis. Widespread polymorphism was found for three of the four components of the two genetic systems responsible for the two barriers, with at least 13 populations possessing genes for one or both of the barriers. In one system (the C7/U8 system; Christie and Macnair, 1984), the distribution of the two component genes was correlated with the morphometrical similarity and geographical location of the populations. This pattern could be produced by a historical association or by an adaptive response. In the other system (the Cerig/C10 system; Macnair and Christie, 1983), the genes were more widely dispersed, and there was no obvious morphometrical or geographical association. Populations possessing the complementary factors causing partial reproductive isolation are not always widely separated geographically. These results indicate that the spread of postmating reproductive isolating genes through drift, selection, or hitchhiking could readily cause reproductive isolation to evolve in this species.>     

THE EFFECTS OF FIVE GENERATIONS OF ENFORCED SELFING ON POTENTIAL MALE AND FEMALE FUNCTION IN MIMULUS GUTTATUS

In prior work we detected no significant inbreeding depression for pollen and ovule production in the highly selfing Mimulus micranthus, but both characters showed high inbreeding depression in the mixed-mating M. guttatus. The goal of this study was to determine if the genetic load for these traits in M. guttatus could be purged in a program of enforced selfing. These characters should have been under much stronger selection in our artificial breeding program than previously reported characters such as biomass and total flower production because, for example, plants unable to produce viable pollen could not contribute to future generations. Purging of genetic load was investigated at the level of both the population and the individual maternal line within two populations of M. guttatus. Mean ovule number, pollen number, and pollen viability declined significantly as plants became more inbred. The mean performance of outcross progeny generated from crosses between pairs of maternal inbred lines always exceeded that of self progeny and was fairly constant for each trait through all five generations. The consistent performance of outcross progeny and the universally negative relationships between performance and degree of inbreeding are interpreted as evidence for the weakness of selection relative to the quick fixation of deleterious alleles due to drift during the inbreeding process. The selective removal (purging) of deleterious alleles from our population would have been revealed by an increase in performance of outcross progeny or an attenuation of the effects of increasing homozygosity. The relationships between the mean of each of these traits and the expected inbreeding coefficient were linear, but one population displayed a significant negative curvilinear relationship between the log of male fertility (a function of pollen number and viability) and the inbreeding coefficient. The generally linear form of the responses to inbreeding were taken as evidence consistent with an additive model of gene action, but the negative curvilinear relationship between male fertility and the inbreeding coefficient suggested reinforcing epistasis. Within both populations there was significant genetic variation among maternal lineages for the response to inbreeding in all traits. Although all inbred lineages declined at least somewhat in performance, several maternal lines maintained levels of performance just below outcross means even after four or five generations of selfing. We suggest that selection among maternal lines will have a greater effect than selecting within lines in lowering the genetic load of populations.     

RESPONSE TO NATURAL ENVIRONMENTAL HETEROGENEITY: MATERNAL EFFECTS AND SELECTION ON LIFE-HISTORY CHARACTERS AND PLASTICITIES IN MIMULUS GUTTATUS

Recent studies in plant populations have found that environmental heterogeneity and phenotypic selection vary at local spatial scales. In this study, I ask if there is evolutionary change in response to environmental heterogeneity and, if so, whether the response occurs for characters or character plasticities. I used vegetative clones of Mimulus guttatus to create replicate populations of 75 genotypes. These populations were planted into the natural habitat where they differed in mean growth, flowering phenology, and life span. This phenotypic variation was used to define selective environments. There was variation in fitness (flower production) among genotypes across all planting sites and in genotype response to the selective environment. Offspring from each site were grown in the greenhouse in two water treatments. Because each population initially had the same genetic composition, variation in the progeny between selective environments reveals either evolutionary change in response to environmental heterogeneity or environmental maternal effects. Plants from experimental sites that flowered earlier, had shorter life spans and were less productive, produced offspring that had more flowers, on average, and were less plastic in vegetative allocation than offspring of longer-lived plants from high-productivity areas. However, environmental maternal effects masked phenotypic differences in flower production. Therefore, although there was evidence of genetic differentiation in both life-history characters and their plasticities in response to small-scale environmental heterogeneity, environmental maternal effects may slow evolutionary change. Response to local-scale selective regimes suggests that environmental heterogeneity and local variation in phenotypic selection may act to maintain genetic variation.