David E. Carr

FIVE GENERATIONS OF ENFORCED SELFING AND OUTCROSSING IN MIMULUS GUTTATUS: INBREEDING DEPRESSION VARIATION AT THE POPULATION AND FAMILY-LEVEL

The focus of this study was to examine the consequences of five sequential generations of enforced selfing and outcrossing in two annual populations of the mixed‐mating Mimulus guttatus. Our primary goal was to determine whether purging of deleterious recessive alleles occurs uniformly between populations and among families, and thus gain insights into the mode of gene action (dominance, overdominance, and/or epistasis) governing the expression of inbreeding depression at both the population and family levels across the life cycle.

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

Abstract 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 aboveground 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.

Genetics underlying inbreeding depression in Mimulus with contrasting mating systems

The importance of inbreeding depression in theoretical considerations of mating-system evolution and its potential impact on the persistence of small populations has renewed interest in the genetic basis of this phenomenon. Inbreeding increases homozygosity. This can produce inbreeding depression for two different reasons: first, deleterious recessive or partially recessive alleles that are masked at heterozygous loci by dominant alleles become fully expressed in homozygotes; and second, alleles may interact in an overdominant manner, such that the fitness of either type of homozygote is lower than that of heterozygotes. These two mechanisms produce different long-term effects in populations experiencing increased levels of inbreeding. Inbreeding depression resulting from deleterious alleles can be removed by selection, but inbreeding depression produced by overdominance cannot be removed without lowering the mean fitness of the population. Using a North Carolina 3 breeding programme, the most powerful quantitative genetics technique available, we show here that deleterious recessive alleles are mainly responsible for inbreeding depression in two closely related annual plants, the primarily selfing Mimulus micranthus and the mixed-mating M. guttatus. Estimates indicate that deleterious alleles in M. micranthus are more nearly additive than they are in M. guttatus.

Inbreeding depression under a competitive regime in Mimulus guttatus : consequences for potential male and female function

The effects of one generation of selfing and outcrossing on six life history characters were examined in the greenhouse for two populations of the mixed-mating species Mimulus guttatus. Inbreeding depression was not detected in the mass of seed produced by maternal plants, but selfing significantly reduced germination success by 13–20 per cent relative to outcross seeds. Seedlings produced by self-pollination were 26–31 per cent smaller than outcross progeny. Late life history stages were examined under varied competitive regimes in which each plant was grown with a neighbour of either an inbred or outbred individual. Progeny resulting from self-pollinations showed a 14–30 per cent reduction in ovule number per locule, and their pollen production per flower was reduced by 28–33 per cent. Adult above-ground biomass showed the greatest inbreeding depression, ranging from 24–50 per cent depending on the competitive conditions. Thus progeny resulting from selfing are smaller as adults and contribute significantly fewer potential gametes into the gene pool as pollen grains and ovules. The multiplicative effects of these characters is calculated. For all traits except ovule number, outcross progeny from parents of the same population did not differ significantly from outcross progeny produced by parents that came from populations separated by 10 km, suggesting relatively little differentiation between populations. The relative performance of self and outcross progeny in later life history stages was independent of the relative performance observed in earlier stages.

EFFECTS OF INBREEDING IN MIMULUS GUTTATUS ON TOLERANCE TO HERBIVORY IN NATURAL ENVIRONMENTS

Inbreeding, which is common in plants, may increase the vulnerability of populations to natural enemies. Similarly, natural enemies may increase the expression of inbreeding depression in their hosts, resulting in altered selection on host mating-system evolution. To examine effects of inbreeding on tolerance to herbivory, we transplanted experimentally self- and cross-fertilized plants into four field populations of Mimulus gut- tatus and applied single Philaenus spumarius (spittlebug) nymphs to half. At the end of the growing season, we scored plants for five fitness components (reproductive effort, biomass, survival, probability of producing flowers or buds, and probability of bolting). Inbreeding reduced population-level tolerance to spittlebug herbivory with respect to plant aboveground biomass. Inbreeding effects on tolerance varied significantly among plant families for three fitness traits, indicating the opportunity for selection by herbivores to improve tolerance in inbreeding populations. These results also indicate that herbivores can alter inbreeding depression in plants. Our results mirror earlier greenhouse studies of inbreeding effects on plant-herbivore interactions, and demonstrate that these effects can be manifested in natural settings as well. This study indicates that inbreeding in natural populations can affect fitness not only directly, but also indirectly through altered inter- actions with natural enemies.

Effects of herbivory and inbreeding on the pollinators and mating system of Mimulus guttatus (Phrymaceae)

Most models of mating system evolution predict mixed mating to be unstable, although it is commonly reported from nature. Ecological interactions with mutualistic pollinators can help account for this discrepancy, but antagonists such as herbivores are also likely to play a role. In addition, inbreeding can alter ecological interactions and directly affect selfing rates, which may also contribute to maintaining mating system variation. We explored herbivore and inbreeding effects on pollinator behavior and selfing rates in Mimulus guttatus. First, individual spittlebug (Philaenus spumarius) herbivores were applied to native plants in two populations. Spittlebugs reduced flower size, increased anther-stigma distance, and increased selfing rates. A second experiment factorially crossed spittlebug treatment with inbreeding history (self- vs. cross-fertilized), using potted plants in arrays. Spittlebugs did not affect pollinator behavior, but they reduced flower size and nearly doubled the selfing rate. Inbreeding reduced the frequency of pollinator visits and increased flower-handling time, and this may be the first report that inbreeding affects pollinator behavior. Selfing rates of inbred plants were reduced by one half, which may reflect early inbreeding depression or altered pollinator behavior. The contrasting effects of herbivory and inbreeding on selfing rates may help maintain mating system variation in M. guttatus.

The molecular clock and the relationship between population size and generation time

A rule of thumb in population genetics is that population size (N) and generation time (g) across taxa are inversely related. This relationship played a prominent role in the development of the neutral theory of molecular evolution because it was used by Ohta to explain why a molecular clock is compatible with the theory (Ohta, 1977, 1987; Kimura, 1979, 1983; Ohta and Tachida, 1990). However, such a relationship has never been documented. Nei and Graur (1984) failed to find a significant correlation in the only known attempt to test the relationship. They did not, however, test for a correlation between log(N) and log(g), which is the relationship required to produce a molecular clock in Ohtas original model (Ohta, 1977; Kimura, 1979, 1983). We have reanalyzed Nei and Graurs data set and report a significant negative correlation between log(N) and log(g).

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.

Tests for the joint evolution of mating system and drought escape in Mimulus

BACKGROUND AND AIMS Self-fertilizing taxa are often found at the range margins of their progenitors, where sub-optimal habitats may select for alternative physiological strategies. The extent to which self-fertilization is favoured directly vs. arising indirectly through correlations with other adaptive life history traits is unclear. Trait responses to selection depend on genetic variation and covariation, as well as phenotypic and genetic responses to altered environmental conditions. We tested predictions of the hypothesis that self-fertilization in Mimulus arises through direct selection on physiological and developmental traits that allow seasonal drought escape. METHODS Phenotypic selection on mating system and drought escape traits was estimated in field populations of M. guttatus. In addition, trait phenotype and phenotypic selection were compared between experimental wet and dry soil in two greenhouse populations each of M. guttatus and M. nasutus. Finally, genetic variation and covariation for traits were compared between wet and dry soil treatments in a greenhouse population of M. guttatus. KEY RESULTS Consistent with predictions, selection for early flowering was generally stronger than for mating system traits, and selection for early flowering was stronger in dry soil. Inconsistent with predictions, selection for water-use efficiency was largely absent; selection for large flowers was stronger than for drought escape in the field; and most drought escape and mating system traits were not genetically correlated. A positive genetic correlation between flowering time and flower size, which opposed the adaptive contour, emerged only in wet soil, suggesting that variation in water availability may maintain variation in these traits. Plastic responses to soil moisture treatments supported the idea that taxonomic divergence could have been facilitated by plasticity in flowering time and selfing. CONCLUSIONS The hypothesis that plant mating systems may evolve indirectly via selection on correlated life history characteristics is plausible and warrants increased attention.

The susceptibility and response of inbred and outbred Mimulus guttatus to infection by Cucumber mosaic virus

The deleterious effects of inbreeding have been well documented, but only recently have studies begun to explore the consequences of inbreeding for important ecological interactions. We examined the effects of inbreeding on the interaction between host and pathogen using the mixed-mating Mimulus guttatus (Scrophulariaceae) and Cucumber mosaic virus (CMV). Inbred (self) and outbred M. guttatus from two California populations (M5 and M13) were rub-inoculated with CMV and compared to sham-inoculated controls. Flower production by outbred plants in host population M5 showed little effect of the inoculation treatment, but inoculation reduced flower production of inbred plants by 12%, indicating that inbreeding reduces tolerance to CMV infection. This interaction fell short of significance, however. The effects of inbreeding and CMV inoculation on biomass in M5 varied significantly across the 15 families used in this experiment, indicating genetic variation in the effect of inbreeding on resistance or tolerance to CMV. CMV infection reduced biomass in host population M13, but there were no significant interactions between virus treatment and level of inbreeding for either flower production or biomass. Enzyme linked immunosorbent assay (ELISA) was used to detect CMV in host tissues. In both populations, mean ELISA absorbance values of inoculated plants were nearly identical for self and outcross hosts, indicating equal susceptibility to CMV. In outbred plants of population M5, flower production did not change with increasing ELISA absorbance, but in inbred plants it declined, indicating reduced tolerance to CMV infection. The results from this study suggest that pathogens may become increasingly detrimental as host populations become more inbred.