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.     

Analysis of the relationship between allozyme heterozygosity and fitness in the rare Gentiana pneumonanthe L.

Especially for rare species occurring in small populations, which are prone to loss of genetic variation and inbreeding, detailed knowledge of the relationship between heterozygosity and fitness is generally lacking. After reporting on allozyme variation and fitness in relation to population size in the rare plant Gentiana pneumonanthe, we present a more detailed analysis of the association between heterozygosity and individual fitness. The aim of this study was to test whether increased fitness of more heterozygous individuals is explained best by the ‘inbreeding’ hypothesis or by the ‘overdominance’ hypothesis. Individual fitness was measured during 8 months of growth in the greenhouse as the performance for six life-history parameters. PCA reduced these parameters to four main Fitness Components. Individual heterozygosity was scored for seven polymorphic allozyme loci. For some of these loci (e.g. Aat3, Pgm1 and 6Pgdh2) heterozygotes showed a significantly higher relative fitness than homozygotes. To test the inbreeding model, regression analyses were performed between each Fitness Component and the number of heterozygous loci per individual. Multiple regressions with the adaptive distance of five loci as independent variables were used to test the overdominance model. Only the inbreeding model was a statistically significant explanation for the relationship between heterozygosity and fitness in G. pneumonanthe. The number of heterozygous loci was significantly negatively correlated with the coefficients of variation of three of the six initially measured fitness parameters. This suggests a lower developmental stability among more homozygous plants and may explain the higher phenotypic variation in small populations of the species observed earlier. The importance of the results for conservation biology is discussed.     

Microsatellite variation and evolution in the Mimulus guttatus species complex with contrasting mating systems

Mutational variability at microsatellite loci is shaped by both population history and the mating system. In turn, alternate mating systems in flowering plants can resolve aspects of microsatellite loci evolution. Five species of yellow monkeyflowers (Mimulus sect. Simiolis) differing for historical rates of inbreeding were surveyed for variation at six microsatellite loci. High levels of diversity at these loci were found in both outcrossing and selfing taxa. In line with allozyme studies, inbreeders showed more partitioning of diversity among populations, and diversity in selfing taxa was lower than expected from reductions in effective population size due to selfing alone, suggesting the presence of either population bottlenecks or background selection in selfers. Evaluation of the stepwise mutation model (a model of DNA replication slippage) suggests that these loci evolve in a stepwise fashion. Inferred coalescent times of microsatellite alleles indicate that past bottlenecks of population size or colonization events were important in reducing diversity in the inbreeding taxon.      

Genotypic selection in Daphnia populations consisting of inbred sibships

The genetic basis of fitness reduction associated with inbreeding is still poorly understood. Here we use associations between allozyme genotypes and fitness to investigate the genetic basis of inbreeding depression in experimental outdoor populations of the water flea, Daphnia magna. In Daphnia, a phase of clonal reproduction follows hatching from sexually produced resting eggs, and changes in genotype frequencies during the clonal phase can be used to estimate fitness. Our experiment resembles natural colonization of ponds in that single clones colonize an empty pool, expand asexually and produce sexual offspring by selfing (sisters mate with their clonal brothers). These offspring diapause and form populations consisting of selfed sibships in the following spring. In 12 of 13 experimental populations, genotypes of selfed hatchlings after diapause conformed to Mendelian expectations. During the subsequent ca. 10 asexual generations, however, genotype frequencies changed significantly at 19 of 27 single loci studied within populations, mostly in favour of heterozygotes, with heterozygosity at multiple loci affecting the change in genotype frequency multiplicatively. Because variance in heterozygosity among siblings at a given marker reflects only heterozygosity in the chromosomal region around this marker, our results suggest that selection at fitness-associated loci in the chromosomal regions near the markers were responsible for these changes. The genotype frequency changes were more consistent with selection acting on linked loci than on the allozymes themselves. Taken together, the evidence for abundant selection in the chromosomal regions of the markers and the fact that changes in genotype frequencies became apparent only after several generations of clonal selection, point to a genetic load consisting of many alleles of small or intermediate effects, which is consistent with the strong genetic differentiation and repeated genetic bottlenecks in the metapopulation from which the animals for this study were obtained.     

Effect of mating structure on variation in inbreeding

An analysis is made of the variation among individuals in finite populations of the proportion of their genes which are identical by descent. There are two causes of this variability: variation in pedigree among individuals, and linkage which causes whole blocks of genes to be identical or nonidentical by descent. The variation between and within populations is analyzed in detail for several mating systems: monoecious populations with and without random selfing, and dioecious populations with and without a hierarchical mating structure. Transition matrices for two-locus descent measures are given for each system. Total variability is obtained by integrating these measures over the distribution of map distances over whole chromosomes. Approximate methods are also developed for unlinked loci. Unless populations have a very small effective size (N_e) there is little variation in inbreeding between populations. For unlinked loci, the coefficient of variation in nonidentity within populations approaches about 1/√3N_e for random selfing, 1/√6N_e for monogamous matings and 1/√12N_e for monoecy with selfing excluded or dioecy with random pairing. If there is no association between map distance and initial heterozygosity or effect on quantitative traits, the coefficient of variation in mean heterozygosity over the genome is related to that in nonidentity, and the additional variation in a quantitative trait due to dominant genes equals the product of the square of the initial inbreeding depression and the squared coefficient of variation of nonidentity.     

Genetic variation and the mating system in the rare Acacia sciophanes compared with its common sister species Acacia anfractuosa (Mimosaceae)

Acacia sciophanes is an extremely rare and Critically Endangered species known from two small populations separated by less than 7 km. Specifically we aimed to investigate whether rarity in A. sciophanes is associated with decreased levels of genetic variation and increased levels of selfing by comparing patterns of genetic variation and mating system parameters with its widespread and common sister species A. anfractuosa. Fourteen polymorphic allozyme loci were used to assess genetic diversity with four of these used in the estimation of mating system parameters. At the species level A. sciophanes has lower allelic richness, polymorphism, observed heterozygosity and gene diversity than A. anfractuosa and significantly lower levels of gene diversity at the population level. Both species have a mixed mating system but the largest population of A. sciophanes has lower levels of outcrossing, higher correlated paternity and increased bi-parental inbreeding compared with two A. anfractuosa populations. However, both correlated paternity and bi-parental inbreeding appear to be at least partly influenced by population size regardless of the species. We suggest that A. sciophanes is likely to be an intrinsically rare species and that in particular the lower levels of genetic diversity and increased selfing are a feature of a species that has the ability to persist in a few localised small populations. Despite recent extensive habitat destruction our comparative study provided no clear evidence that such events have contributed to the lower genetic diversity and increased selfing in A. sciophanes and we believe its ability to exist in small populations may not only be an important factor in its survival as a rare species but also indicates that it may be less susceptible to the impacts of habitat loss and fragmentation. The key to this species conservation will be the maintenance of suitable habitat, particularly through improved fire regimes and control of invasive weeds, that will allow the two small populations to continue to persist in extremely restricted areas of remnant vegetation.     

Self-fertilization in mosses: a comparison of heterozygote deficiency between species with combined versus separate sexes

Self-fertilization is a key difference of adaptive significance between species with combined versus separate sexes. In haploid-dominant species such as mosses and ferns, species with either combined or separate sexes (monoicous and dioicous, respectively) have the potential to self-fertilize (intergametophytic selfing), but being monoicous allows an additional mode of selfing (intragametophytic selfing). We used allozyme electrophoresis to estimate deviations from expected levels of heterozygosity under Hardy-Weinberg equilibrium to infer selfing rates in 10 moss species from 36 New Zealand populations. We found that while there were deficiencies of heterozygotes compared to expectation in both monoicous and dioicous mosses, monoicous species had significantly higher levels of heterozygote deficiency than dioicous species (F(IS)=0.89+/-0.12 and 0.41+/-0.11, respectively). Estimated selfing rates suggest that selfing occurs frequently in monoicous populations, and rarely in dioicous populations. However, in two dioicous species (Polytrichadelphus magellanicus and Breutelia pendula), we found significant indications of mixed mating or biparental inbreeding in a handful of populations. These data provide the first analysis of heterozygote deficiency and selfing among haploid-dominant species with breeding system variation, and we discuss our results with respect to the consequences of inbreeding depression and the evolution of breeding systems.     

Self-fertilization in the white-lipped land snail Triodopsis Albolabris

The breeding system of the polygyrid land snail Triodopsis albolabris was studied in laboratory colonies through the use of allozyme-genetic markers. Isolated virgin individuals self-fertilized only after several months of apparent self-sterility. Isolated pairs appeared to reproduce solely by outcrossing. Overall, the normalized reproductive success of paired individuals was about 86 times greater than that of isolates.
Nine natural populations of this snail were surveyed at eight allozyme loci. All populations were highly polymorphic, and heterozygosity was high and agreed with Hardy-Weinberg expectations. This indicates that selfing is not common in established populations. We conclude that T. albolabris avoid inbreeding by selfing if at all possible. However, if the probability of finding a mate is low (assessed by several months without finding one), lone individuals self-fertilize.     

INBREEDING DEPRESSION IN FOUR POPULATIONS OF COLLINSIA HETEROPHYLLA NUTT (SCROPHULARIACEAE)

The effects of one and two generations of inbreeding were studied in plants from four natural populations of the annual plant, Collinsia heterophylla, using inbred and outcrossed plants generated by hand pollinations to create expected inbreeding coefficients ranging from 0–0.75. The selfing rates of the populations were estimated using allozyme markers to range from 0.37–0.69. Inbreeding depression was mild, ranging from 5–40%, but significant effects were detected for characters measured at all stages of the life cycle. Fitness components declined significantly with the inbreeding coefficient, and regression of fitness characters on inbreeding coefficients gave no evidence of any strongly synergistic effects attributable to the different genetic factors that contribute to decline in fitness under inbreeding. The magnitude of inbreeding depression did not clearly decrease with the populations' levels of inbreeding. This is not surprising because the selfing rates are similar enough that it is unlikely that the populations have been characterized for long periods of time by these different inbreeding levels.     

Growth performance of hybrid families by crossing selfed lines of Betula pendula Roth

Hybrid breeding is an effective approach in many agricultural crops. In allogamous tree species severe inbreeding depression and long reproductive cycles generally prohibit its use. However, three generations of selfing in silver birch (Betula pendula Roth) were obtained by forcing trees to flowering under greenhouse conditions. Hybrids were produced by crossing first-, second and third-generation selfed lines. The effects of different levels of parental inbreeding on the growth performance of hybrid families were observed in a 9-year-old field progeny test. Also, provenance crosses were carried out between selfed lines from different parts of Finland and several other European countries. Observations of growth performance of the provenance hybrids were made in the same trial. The results indicated that the mean stem volumes were significantly different between classes of parental in breeding coefficients (F_P) (P<0.0001), and were positively correlated with F_P (r=0.9106, P<0.05). Within-family variation of the hybrid families decreased with an increase of F_P. The performance of the provenance crosses between parents at a relatively close distance did not depart significantly from the standard controls. However, when the cross distance was extended far to the south, hybrids grew faster, indicating either higher heterozygosity or an extended growth period.     

Allozyme variation in Anopheles stephensi Liston from Pakistan (Diptera: Culicidae)

Allozyme variability was analyzed at 16 loci in 11 lines of Anopheles stephensi Liston from Pakistan. Six lines were considered as samples from natural populations. For these lines the mean number of alleles was 1.31-1.63, the degree of polymorphism was 0.188-0.375, the observed heterozygosity was 0.065-0.086, and the genetic distance ranged from 0.001 to 0.016. No population-specific alleles were found. Interbreeding was considerable (mean Fit = 0.183). Differences in allele frequencies were due considerable (mean Fit = 0.183). Differences in allele frequencies were due primarily to local inbreeding (Fis greater than Fst at most loci). The Lahore line, reared for more than 20 generations, had more homozygotes than the other lines. A line refractory to Plasmodium falciparum and a genetic sexing line exhibited decreased allozyme variability. The latter line showed reduced staining intensity at 10 loci. Linkage studies are recommended for the following loci with rare alleles: Acp, Gapdh, Icd-1, Icd-2, Mpi, and Pgd.     

Trends in heterozygosity in the process of producing inbred strains of Japanese quail

Trends in heterozygosity in the process of producing inbred strains of Japanese quail were examined through the characterization of protein polymorphisms based on gene frequencies of 7 polymorphic loci. The average heterozygosity ( H _o) at generation 1 was 0.472 and it decreased with increasing inbreeding coefficient (F) to 0.214 at generation 5 when F was 0.594. In all generations, the observed heterozygosities of the surviving families tended to be higher than those of the families that did not survive. The frequency of heterozygotes of the Es-4 locus in surviving families was higher than that of the extinct families in each generation and the difference became conspisuous in generation 4. These results suggests that a heterozygote advantage of Es-4 locus is revealed by inbreeding.     

Heterosis for neonatal survival in the guppy

Neonatal survival rate ranged from 91.8 to 100.0% in 12 populations of the guppy Poecilia reticulata , and was higher in naturalized Japanese stream populations than in domestic strains. Mean heterozygosity at five allozyme and four microsatellite loci varied between 0.112 and 0.430 and was significantly correlated with neonatal survival rate among populations, suggest ing that inbreeding decreases neonatal survival. Diallel and reciprocal crosses among four domestic strains demonstrated heterosis for neonatal survival. These results indicate that neonatal survival is genetically affected by heterosis and its antithesis, inbreeding depression. The relationship between neonatal survival and the mean heterozygosity suggests that overall heterozygosity is important for neonatal survival of the guppy.     

Evolutionary implications of a high selfing rate in the freshwater snail Lymnaea truncatula

Abstract Self-compatible hermaphroditic organisms that mix self-fertilization and outcrossing are of great interest for investigating the evolution of mating systems. We investigate the evolution of selfing in Lymnaea truncatula , a self-compatible hermaphroditic freshwater snail. We first analyze the consequences of selfing in terms of genetic variability within and among populations and then investigate how these consequences along with the species ecology (harshness of the habitat and parasitism) might govern the evolution of selfing. Snails from 13 localities (classified as temporary or permanent depending on their water availability) were sampled in western Switzerland and genotyped for seven microsatellite loci. F_IS (estimated on adults) and progeny array analyses (on hatchlings) provided similar selfing rate estimates of 80%. Populations presented a low polymorphism and were highly differentiated (F_ST= 0.58). Although the reproductive assurance hypothesis would predict higher selfing rate in temporary populations, no difference in selfing level was observed between temporary and permanent populations. However, allelic richness and gene diversity declined in temporary habitats, presumably reflecting drift. Infection levels varied but were not simply related to either estimated population selfing rate or to differences in heterozygosity. These findings and the similar selfing rates estimated for hatchlings and adults suggest that within-population inbreeding depression is low in L. truncatula.     

Population genetic structure and differentiation of Anthoxanthum alpinum in the subalpine-alpine ecocline of Swiss Alps

Allozyme variation in 6 enzyme systems coding 10 loci was estimated for 18 subpopulations of Anthoxanthum alpinum from three altitudinal transects in two localities of the Swiss Alps. Mean proportions of polymorphic loci (95% criterion), average number of alleles per locus, and mean expected heterozygosity were 64.9%, 2.37 and 0. 252, respectively. Mean genetic distance among populations was 0.011, and 79% of the genetic variation resided within populations. Based on allozyme analysis, marginal subpopulations appeared to have similar level of genetic variability to central subpopulations. Relatively high genetic differentiation, low gene flow values and small neighbourhood sizes suggested that inbreeding followed by genetic drift was possible causes of lowgenetic variability in Arpette A. alpinum populations.     

The evolution and maintenance of monoecy and dioecy in Sagittaria latifolia (Alismataceae)

Plant species rarely exhibit both monoecious and dioecious sexual systems. This limits opportunities to investigate the consequences of combined versus separate sex function on mating patterns and genetic variation and the analysis of factors responsible for the evolution and maintenance of the two sexual systems. Populations of the North American clonal aquatic Sagittaria latifolia are usually either monoecious or dioecious and often grow in close geographic proximity. We investigated mating patterns, genetic structure, and relationships between the two sexual systems using allozyme variation in populations from southern Ontario, Canada. As predicted, selfing rates in monoecious populations (n = 6, mean = 0.41) were significantly higher than in dioecious populations (n = 6, mean = 0.11). Moreover, marker-based estimates of inbreeding depression (delta) indicated strong selection against inbred offspring in both monoecious (mean delta = 0.83) and dioecious (mean delta = 0.84) populations. However, the difference in selfing rate between the sexual systems was not reflected in contrasting levels of genetic variation. Our surveys of 12 loci in 15 monoecious and 11 dioecious populations revealed no significant differences in the proportion of polymorphic loci (P), number of alleles per locus (A), or observed and expected heterozygosity (H(o) and H(e), respectively). Strong inbreeding depression favoring survival of outcrossed offspring may act to maintain similar levels of diversity between monoecious and dioecious populations. Despite geographical overlap between the two sexual systems in southern Ontario, a dendrogram of genetic relationships indicated two distinct clusters of populations largely corresponding to monoecious and dioecious populations. Reproductive isolation between monoecious and dioecious populations appears to be governed, in part, by observed differences in habitat and life history. We suggest that selfing and inbreeding depression in monoecious populations are important in the transition from monoecy to dioecy and that the maintenance of distinct sexual systems in S. latifolia is governed by interactions between ecology, life history, and mating.     

Maintenance of Inbreeding Depression in a Highly Self-Fertilizing Tree, Magnolia obovata Thunb

Inbreeding depression is a major selective force that maintains outcrossing in flowering plants. If the long life and large mature size of trees cause high inbreeding depression via mitotic mutations and half-sib competition, these characteristics may increase inbreeding depression sufficiently to maintain traits that facilitate outcrossing even with high primary selfing rates (proportion of selfed ovules). Here, I report the maintenance of inbreeding depression in a population of a tree (Magnolia obovata Thunb.) with primary selfing rates greater than 0.8 resulting from geitonogamy. The progenies exhibited inbreeding depression for germination, seedling survival, and seedling mass (δ = 0.29–0.38), but no significant difference between crossing type in seedling height. Cumulative inbreeding depression for early survival (from zygote to 2-year-old stage) estimated from these results and from prior data on embryonic survival was high (δ_e = 0.91). The fixation index at maturity based on six allozyme loci was low (F_is = 0.08), indicating that significant inbreeding depression for late survival results in a low level of inbreeding with respect to gene transmission to the next generation. From these results, I estimated that inbreeding depression for late and lifetime survival equaled 0.69 and 0.97, respectively. These results suggest that M. obovata trees maintain high inbreeding depression at both early and late life stages, resulting in a low level of inbreeding despite a high primary selfing rate. The high inbreeding depression can be explained by previous theories and is consistent with the predicted maintenance of inbreeding depression in highly self-fertilizing tree populations. The inbreeding load due to the high primary selfing rate represents a cost of this tree’s pollination system for outcrossing, which is based on automimicry and mass flowering. Co-ordinating editor: S.-M. Chang     

Combined effects of bottlenecks and selfing in populations of Corella eumyota, a recently introduced sea squirt in the English Channel

The success of an exotic species depends notably on its capacity to initiate a new population from a few individuals, to survive genetic bottlenecks and to adapt locally. Species with multiple reproductive strategies (e.g. mixed-mating system with both self- and cross-fertilization) can be efficient colonizers. Herein we focus on Corella eumyota , an exotic ascidian that has rapidly invaded English Channel coasts in recent years. Interestingly, this brooding hermaphroditic ascidian is capable of self-fertilization in the laboratory. We developed 12 microsatellite markers from an enriched library of genomic DNA to investigate the level of inbreeding and selfing in two putatively native populations (South Africa, N  = 34, and New Zealand, N  = 28) and to examine if founder effects were possibly associated with its recent introduction in two French populations (Perros-Guirec, N  = 22 and Brest; N  = 25). Genetic polymorphism was very low in both native populations (i.e. less than 60% of the loci were polymorphic) and even lower in the introduced populations, one of which was monomorphic at all loci, suggesting a recent bottleneck. F _is and a new method based on multi-locus heterozygosity were used to provide estimates of inbreeding. A high selfing rate was inferred in the South Africa population with both methods ( s  = 0.90), whereas in the other native population (New Zealand) a lower but significant estimate of selfing rate ( s  = 0.29) was obtained with the multi-locus method. This variability of population selfing rate might be explained by a mixed-mating system, allowing C. eumyota to reproduce through inbreeding and outbreeding according to mating possibilities; this trait may have favoured the rapid establishment of new populations in Europe.     

INBREEDING DEPRESSION IN TWO MIMULUS TAXA MEASURED BY MULTIGENERATIONAL CHANGES IN THE INBREEDING COEFFICIENT

In mixed-mating plant populations, one can estimate the relative fitness of selfed progeny w by measuring the inbreeding coefficient F and selfing rate s of adults of one generation, together with F of adults in the following generation (after selection). In the first application of this multigenerational method, we estimated F and s for adults over three consecutive generations in adjacent populations of two annual Mimulus taxa: the outbreeding M. guttatus and the inbreeding M. platycalyx. This gave estimates of w for the last two generations. Although average multilocus selfing rates were high in both taxa (0.63 in M. guttatus; 0.84 in M. platycalyx), the relative fitness of selfed progeny averaged only 0.19 in M. guttatus and 0.32 in M. platycalyx. An alternative estimator for w that incorporates biparental inbreeding gave even lower estimates of w. These values are significantly below the 0.5 threshold thought to favor selfing, and show that partially selfing populations can harbor substantial genetic load. In accordance with the purging hypothesis, the more highly selfing M. platycalyx showed marginally lower inbreeding depression than M. guttatus in both years (P = 0.08). Inbreeding depression and selfing rates also varied among years in concert among taxa. Several sources of bias are discussed, but computer simulations indicate it is unlikely that w is biased downwards by linkage of marker loci to load loci.     

Effect of recurrent selfing on inbreeding depression and mating system evolution in an autopolyploid plant

Genome duplication resulting in polyploidy can have significant consequences for the evolution of mating systems. Most theory predicts that self‐fertilization will be selectively favored in polyploids; however, many autopolyploids are outcrossing or mixed‐mating. Here, we examine the hypothesis that the evolution of selfing is restricted in autopolyploids because the genetic cost of selfing (i.e., inbreeding depression) increases monotonically with successive generations of inbreeding. Using the herbaceous, autotetraploid plant Chamerion angustifolium, we generated populations with different inbreeding coefficients (F= 0, 0.17 and 0.36) through three consecutive generations of selfing and compared their magnitudes of inbreeding depression in a common environment. Mating system estimates for four natural populations confirmed that tetraploid selfing rates (s_m= 0.25, SE = 0.02) are similar to those of diploids (s_m= 0.12, SE = 0.12; F_1,2= 1.34, P= 0.37) indicating that both cytotypes are predominantly outcrossing. Compared to an outbred control line, mean inbreeding depression for seed production, survival, and height (vegetative and total) in the inbred line differed among generations (inbreeding coefficients). Across all stages, inbreeding depression (relative to control) was positively related to generation (inbreeding coefficient). Although the initial costs of inbreeding in extant and newly synthesized polyploids may be low compared to diploids, the monotonic increase in inbreeding depression with repeated inbreeding may limit the extent to which selfing variants are favored.