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Inbreeding depression in four populations of Collinsia heterophylla Nutt (Scrophulariaceae).

Inbreeding depression is the reduction in fitness experienced by the progeny of closely related versus unrelated individuals. It is thought to be a potentially powerful force in the evolution of breeding systems and has been suggested as a possible mechanism to oppose the automatic transmission advantage of self-fertilization (Charlesworth 1980; Uyenoyama et al. 1993). Here we examined (1) the relationship of inbreeding depression to the breeding system; (2) the effect of inbreeding on fitness-related characters throughout the life cycle; and (3) the relationship between relative fitness and the inbreeding coefficient.

Inbreeding depression is probably largely caused by the presence in populations of deleterious mutations with low dominance coefficients (Charlesworth and Charlesworth 1987; Fu and Ritland 1993; Johnston and Schoen 1995), although a contribution from loci with overdominance is also possible (Crow 1948; Charlesworth and Charlesworth 1987; Ziehe and Roberds 1989). On either of these genetic hypotheses, it is theoretically expected that lower levels of inbreeding depression should evolve in inbreeding than outbreeding populations (Lande and Schemske 1985; Charlesworth and Charlesworth 1987, 1990; Ziehe and Roberds 1989; Latta and Ritland 1994). Data from crop species support this prediction (Allard 1960), but although many studies have documented inbreeding depression in natural populations of both outcrossing and self-fertilizing gymnosperm and angiosperm species, relatively few have quantified the populations' breeding systems (Charlesworth and Charlesworth 1987). Data on the relationship of inbreeding depression to levels of inbreeding in natural populations are therefore needed. Such data are also important in testing the theoretical prediction that in the absence of pollen discounting, outcrossing populations should have inbreeding depression exceeding one-half (Lloyd 1979; Charlesworth 1980, 1990; Holsinger et al. 1984; Lande and Schemske 1985; Charlesworth and Charlesworth 1990).

To study the relationship between the breeding system and inbreeding depression, it is preferable to compare populations that differ in their breeding systems but are similar in terms of factors such as life history and population size. Comparisons of populations of the same species, or of closely related groups of species, are therefore desirable. A small number of such studies have been reported (reviewed in Barrett and Kohn 1991; Husband and Schemske 1996). Holtsford and Ellstrand (1990) studied several components of fitness in populations of Clarkia tembloriensis, and flower number was studied in Eichhornia paniculata (Toppings and Barrett, as cited in Barrett and Kohn 1991). Both studies showed a decline in inbreeding depression with increased population selfing rates. The genus Mimulus has been the subject of several inbreeding depression studies. In a sympatric population of M. guttatus and M. platycalyx, the more selfing species, M. platycalyx, had less inbreeding depression than the more outcrossing species (Dole and Ritland 1992). Estimates of inbreeding depression in this study were obtained by the marker-based method of Ritland (1990), which may be subject to some error resulting from linkage between neutral marker loci and fitness-determining loci (Charlesworth 1991). Latta and Ritland (1994) examined the relationship between prior inbreeding and inbreeding depression in 15 populations in the M. guttatus complex, and also found a negative relationship between the populations' inbreeding coefficients estimated from their fixation indices, and their inbreeding depression in seedling height, although no relationship was apparent for several other fitness-related characters. A slight, not statistically significant lowering of inbreeding depression was found in a study of nine populations, from three species, of Amsinckia, with selfing rates estimated to range from 0.25 to close to 1 (Johnston and Schoen 1995). Karron (1989) also found no clear relationship between levels of inbreeding depression and breeding system in four species of Astragalus, but he studied only early stages of the progeny life cycle (up to seedling biomass). The breeding systems of the populations studied were not quantified, but are probably similar in the two species studied most thoroughly (A. linifolius and A. lonchocarpus).

In a review of the results on inbreeding depression from both angiosperms and gymnosperms, Husband and Schemske (1996) found evidence that the effect of breeding system may be different for early and late stages of the life cycle. It is clearly important, in any study of inbreeding depression, to study several life-cycle stages. If different stages of the life cycle are affected by independent genes, such that the total effect on fitness is made up of effects at different stages, the effect of inbreeding on fitness could be underestimated, and the effect of changes in breeding system could be unclear. It is of particular interest to know the relative effects of inbreeding on fertility and survival, because effects at these life-cycle stages have different evolutionary consequences. Loss of inbred progeny resulting from low viability of seeds might be quite unimportant if there is competition between seedlings such that inbred progeny are eliminated, but outcrossed progeny survive. If there is a high chance of outcrossed seed production, there may thus be effectively little or no selection against production of some inbred seeds, despite the high levels of inbreeding depression these progeny experience. Inbreeding depression affecting fertility, however, will lead to selection against inbred progeny. Very large effects of inbreeding on fertility have been found in some studies, for instance Johnston's (1992) study of Lobelia cardinalis and L. siphilitica, species with at least 50% selfing in some populations. Data on late stages of the life cycle, and on fertility, are therefore especially needed.

Inbreeding depression can be estimated in different ways (Johnston and Schoen 1994), and it is important to realize that the use to which the data are to be put influences the choice of method. If one is interested in the level of inbreeding depression that would be experienced by a phenotype with a variant selfing rate introduced into a natural population, one needs to compare the effects of selfing and outcrossing on genotypes taken from the population, and these genotypes should have the natural levels of inbreeding. If, on the other hand, one is interested in estimating the genetic loads of populations, and comparing populations with different levels of inbreeding, it is preferable to start with a set of outbred genotypes, otherwise prior inbreeding will mean that the progeny generated by one generation of selfing consists of a set of genotypes with various inbreeding coefficients in excess of 0.5, whereas progeny from outcrossing will have zero, or very low, inbreeding coefficients. If fitness decreases with increased inbreeding coefficient, the effect of inbreeding on fitness will thus be overestimated in a partially inbreeding population. This would tend to minimize the decline in estimated genetic load if one compared outbred with more inbred populations. It is therefore desirable to create individuals with a range of known inbreeding coefficients. Most previous studies of inbreeding depression, other than those of Willis (1993a) and McCall et al. (1994), have used plants of unknown inbreeding coefficient, and thus fall into the first category mentioned above. Our aim here was to include both types of inbreeding-depression estimates described above. We also included many life-cycle stages.

In addition, studies of the relationship between inbreeding coefficient and fitness provide information about the way that different genes affecting fitness interact (Willis 1993a; McCall et al. 1994). If effects of loci are independent, such that fitnesses are multiplicative, increasing inbreeding coefficients (i.e., homozygosity) should produce decreased fitness proportionately, on a logarithmic scale. If, on the other hand, there are nonmultiplicative interactions, the decline in fitness on the log scale should be nonlinear. Synergistic interactions are important in theoretical models of breeding-system evolution (Kondrashov 1985). If synergism occurs, the fitness of genotypes homozygous for many loci will fall below the value predicted from data on genotypes with fewer homozygous loci; thus, data on relative fitness at several levels of inbreeding are valuable in assessing the likelihood of this form of interaction.

We chose the genus Collinsia for study because breeding systems of species in this genus have been stated to range from inbreeding to outbreeding (Garber 1956, 1974). Inbreeding depression in several stages of the life cycle was found in C. verna (Kalisz 1989), a species thought to have a mixed mating system (Greenlee and Rai 1986; Kalisz 1989), although no quantitative estimates of outcrossing are available. We chose to study populations of C. heterophylla, which has been considered a largely outbreeding species (Weil and Allard 1964; Garber 1974). Outcrossing-rate estimates (t) based on visible genetic markers demonstrated breeding-system variation in three populations studied by Well and Allard (t = 0.62-1.00, 1964). Our study of the selfing rates of several populations using allozyme markers, however, shows a mixed mating system with quite high levels of inbreeding in the four populations studied (outcrossing rates ranging from 0.32-0.64, Charlesworth and Mayer 1995).

MATERIALS AND METHODS

Natural History

Collinsia heterophylla (Scrophulariaceae) is a widely distributed native California annual. The species was reported to be diploid by Garber (1956), which was confirmed by our isozyme profiles. Each plant produces approximately 50-100 flowers in the field, and many more in cultivation. The flowers are arranged in whorls on spikes, and are easily emasculated. The corollas are generally bicolored, with a white to pale purple upper lip and a deep purple lower lip, although in one of the populations we studied (the M population, see below), both the upper and lower lips of the corolla were very pale purple.

Collections

Seeds and flowers were collected from more than 50 families from each of four populations in the San Francisco Bay area. The term "families" refers to all seeds collected from a single plant and will be used to describe maternal lineages that originated in the field-collected seeds. We chose to study four populations to investigate interpopulational variation in the selfing rate and response to inbreeding. We hoped to find populations that varied in the outcrossing rate to test the idea that more outcrossing populations should experience greater inbreeding depression than selfing populations. Table 1 shows the localities and estimated population sizes at the time of flowering. Numbers of flowers and inflorescences per plant were recorded at the time they were first visited, and plants were marked for later identification. A single flower per plant was collected into formalin-acetic acid-alcohol (FAA). We measured flower size (floral tube length and petal lobe length), style length, and anther-stigma separation to determine whether there are relationships between these traits and outcrossing rates. For example, populations with higher selfing rates might be expected to have smaller flowers and/or smaller stigma/anther separation. About one month later, fruits were collected from the marked plants, and from other plants. The seeds were counted and weighed.
TABLE 1. Locations of Collinsia heteropylla populations studied.


                                                              Esti
                                                              mated


popula-
Popula-                                         Elevation     tion
tion                    Location                   (m)        size


D          Mount Diablo State Park Mitchell        180         250
            Canyon, Contra Costa County


H          Mount Hamilton. On roadside, Mt.        610         600
            San Antonio Road, 6.0 mi. east
            of furthest east dome of Lick
            Observatory, Santa Clara County


M          Mount Hamilton. On roadside,             30         400
            Highway 230, near [10.sup.88]
            mile sign on south side of road,
            Santa Clara County


T          Mount Tamalpais. Cataract Trail,        300         500
            Marin County


First Greenhouse Generation

The first greenhouse generation of plants was used to provide material for outcrossing-rate estimates and to create selfed and outcrossed lines in each population. Seeds collected in the field were germinated and grown as described by Charlesworth and Mayer (1995). Germination began after four or five days, and was essentially complete by seven days. At least six seedlings per maternal plant from approximately 50 families per population were grown, and leaf samples from each plant were frozen for electrophoretic analysis to estimate the outcrossing rates of the populations (Charlesworth and Mayer 1995).

Two additional individuals from each of the 50 first-generation families per population were grown to reproductive maturity in an insect-free growth room (16 h/daylight, 24 [degrees] C days/18 [degrees] C nights). Thus, a total of about 100 plants from each of the four populations were grown. Two inflorescences were tagged on one plant per family, one for self-pollinations and another for outcross-pollinations. The second plant was retained in case the first plant died, and to provide a second estimate of the autogamy rate within each family. As they came into flower, plants were arbitrarily paired for reciprocal crosses within populations. It was assumed that outcrossed plants were not paired with relatives; for example, that the 50 families from a given population were unrelated. All flowers, including those to be self-pollinated, were emasculated in the bud with forceps before the anthers dehisced. When the style had elongated and the stigma lobes had become reflexed, indicating receptivity, pollen was applied to the stigma with forceps, and the date and nature of the pollination was recorded. Additional pollen was applied on subsequent days if the style remained erect and it did not appear that fertilization had occurred. Pollinated flowers were marked with spots of acrylic paint on the calyxes to indicate the type of pollination. To obtain at least six capsules per cross type, 6-20 flowers were pollinated, depending on the success of the cross. Capsules were collected when mature and just beginning to split open, and the date of collection was recorded. The numbers and total weight of seeds in each capsule were determined. The autogamy rate for five flower whorls per plant was estimated on the bottom five whorls of a single inflorescence, by recording numbers of flowers and fruit set on these unmanipulated flowers.

Second Greenhouse Generation

The plants in the second generation were scored for various life-history traits to estimate relative fitnesses of progeny derived from selfing and outcrossing. When the plants were mature, a new set of pollinations (described below) was performed on the plants, to create a third-generation set of plants with a range of inbreeding coefficients.

The plants for these experiments were grown as follows: for each set of seeds from the first-generation maternal plants, seeds were pooled within full sibships (i.e., seeds produced by selfing were pooled, and also seeds produced by outcrossing), and 10 seeds from each treatment were haphazardly selected and weighed as a group. The seeds were sown on filter paper in Petri dishes and germinated at 4 [degrees] C. Two outcrossed and two selfed progeny plants per maternal family were arbitrarily selected to be grown under controlled conditions in a growth room, under the same conditions as the first greenhouse generation.

Because of the large numbers of families to be grown, seeds from the four populations were sown in four blocks. Some families did not survive transfer to a new greenhouse. We compared the D and H populations in one pair of blocks and the M and T populations in another. The first block consisted of the selfed and outcrossed lines of half the maternal families from the D and H populations (12-15 families each from each population, for a total of 240 plants per block, see below), and the second block was made up of the remaining families from these same populations. We took care to separate the reciprocal pairs into different blocks to avoid crossing them with each other for the next greenhouse generation. The third and fourth blocks were constructed in the same way as the first two blocks, but using the M and T populations.

A set of fitness-related characters, covering all parts of the life cycle, was scored on the second-generation selfed and outcrossed plants. Seed germination was scored daily and all seedlings were transplanted into pots with vermiculite and later into Promix, as for the first greenhouse generation. Date of germination, percentage of germination, and seedling survival 1 wk after transplanting into vermiculite were recorded. Rate of growth of the seedlings, measured in terms of stem height, was monitored weekly for 10 wk. We also recorded the date of first flowering, the number of secondary branches per plant after 10 wk in Promix, and the number of flowers on the main (central) stem. After 10 wk in the growth room, plants were moved into the greenhouse. Autogamy rates for each flower were recorded as described for the first greenhouse generation. Pollen was placed in a drop of aniline blue in lactophenol on microscope slides for estimation of pollen viability (Radford et al. 1974). A minimum of 300 grains per slide was counted. A single flower from each plant was collected into FAA for ovule counts under a dissecting microscope.
TABLE 2. Pollinations performed on second generation of greenhouse
plants.


Maternal                                           Inbreeding
plant         Type of pollination                  coefficient


selfed        self (SS)                                0.75
selfed        unrelated, same pop (SX)(1)              0
crossed       self (XS)                                0.5
crossed       full sib (Xsib)                          0.25
crossed       unrelated, same pop (XX)(1,2)            0
crossed       unrelated, other pop (Xother)(2)         0


1 To compare fertility of outcrosses of selfed and outcrossed
parents.


2 To investigate outbreeding depression or heterosis between
populations.


When plants began flowering, a set of crosses was made to create plants whose expected inbreeding coefficients ranged from 0-0.75, assuming initially outbred parents (Table 2). By raising an initial generation of outcrossed plants, we ensured that the inbreeding coefficients of the parents to be selfed or sib-mated were zero, except for the possibility that the parents for the first-generation outcrosses were related. Crosses between lines that had been crossed in the first generation were avoided in making pollinations among the second-generation plants, to prevent crosses between relatives resulting in higher inbreeding coefficients than planned. Given the fact that the wild populations we studied were not 100% outcrossing, however, the plants generated from two generations of selfing starting from wild-collected seeds probably had an inbreeding coefficient slightly greater than the expected value of 0.75 (see below). Seeds from the pollinations were collected and germinated as described above.

Third Greenhouse Generation

The third-generation plants consisted of individuals from all four populations with inbreeding coefficients ranging from 0-0.75, as just described. Because of the size of the experiment, the third-generation progeny plants were grown in two blocks in the growth room as described above. All the progeny from the D and H populations were grown together in one block, whereas the progeny from the M and T populations were grown in a second block. Ten seeds from each of the six cross types per family generated from the second-generation plants were germinated as for the second-generation seeds. Two plants from each cross type were arbitrarily selected to be grown to reproductive maturity. The same life-history characters as in the second generation were measured, except that plant height was measured only at 1 and 10 wk. To study the relationship between the breeding system and inbreeding depression, we compared populations that were grown in the same block (M versus T, and D versus H). The numbers of families and progeny plants in this generation that survived to flowering were as follows (starting with 2 plants/cross x 6 crosses/family x number of families): population D, 24 families, 245 plants; H, 22 families, 243 plants; M, 30 families, 332 plants; T, 30 families, 347 plants.

Effects of Harsh Conditions

All the measurements described thus far were calculated with plants growing singly in pots under good growing conditions. To examine the effect of harsher conditions on inbreeding depression, we grew spare third-generation seedlings in very small pots with one outcrossed and one selfed half-sib plant per pot (i.e., seedlings from XX and XS pollinations of the same maternal parent). These seedlings were transplanted at the same time as their siblings, to form two pairs of plants from each maternal plant that yielded enough spare seeds (nearly all maternal plants). At 8 wk after planting, that is, before flowering had started, the plants' heights were measured and the aboveground parts were collected for drying, to obtain dry weights.

Data Analysis

Statview[TM] (Abacus Concepts 1991) was used for most of the statistical analyses. The relationship of inbreeding depression to the breeding system was examined by comparing the estimated outcrossing rates, t (Charlesworth and Mayer 1995), to the estimated inbreeding-depression values for the different populations. The outcrossing rates were corrected to account for the early loss of products of selfing as described below. Inbreeding-depression estimates were obtained by multiplying relative fitness values for a subset of the life-history stages chosen to be independent of each other. Using progeny plants of both the second and third generations, these comparisons were made between populations that were grown together in blocks: D and H, and M and T.

For the second-generation plants, the effect of inbreeding on various life-cycle stages was evaluated by comparing outcrossed and selfed progeny of one generation of inbreeding. Because reciprocal crosses were performed in the outcrossed lines, this allowed tests of the significance of the differences using the method of Lynch (1988), which removes effects of maternal-plant identity.

For the third-generation plants, the effects of selfing and outcrossing were compared by paired t-tests within maternal plants, and linear regressions of fitness-related characters on the inbreeding coefficient were performed. Tests of the significance of the linear-regression coefficients of the natural logarithm-transformed fitness estimates on the inbreeding coefficients were obtained by the jackknife procedure (Efron 1982). To test whether fitness decline with increasing inbreeding coefficient was linear on a multiplicative scale, the residuals from the linear regression were tested to see whether they showed any relationship with the inbreeding coefficient. If there were multiplicative interactions between the loci affecting fitness, that is, the fitness effects of these loci are independent, a linear decline with inbreeding depression is expected when fitness is expressed on a logarithmic scale (see above). Synergistic interactions between loci should cause plants with higher inbreeding coefficients to show a tendency to have fitness values below the linear regression line, on a logarithmic scale, and plants with lower F-values should tend to lie above it (see Willis 1993a). These tests were performed using only the within-population results, because heterosis in crosses between the populations (of which there were a few instances in our data) would increase the fitness values of the F = 0 plants, and might obscure the true shape of the relationship.
TABLE 3. Breeding system results on the four populations and
autogamy rates of their progeny in an insect-free greenhouse.
Standard errors are given below the means. The unadjusted
outcrossing rate is designated by t; [S.sub.1] is the selfing rate
estimated from seedlings (1 - t), and So is the zygote-stage
selfing
rate (see the text for explanation). See the text for an
explanation
of autogamy rate measurements.


                                  Population
                      D          H          M          T


Breeding system


t                   0.50       0.64       0.32       0.56
SE                  0.071      0.076      0.146      0.021
[S.sub.1]           0.50       0.36       0.68       0.44
v                   0.914      0.949      1.02       0.850
[S.sub.0]           0.52       0.37       0.69       0.49
Autogamy of         0.255      0.408      0.468      0.464
progeny plants      0.016      0.028      0.033      0.021


RESULTS

Population Selfing Rates

All four populations yielded estimates of selfing significantly greater than 0 (Charlesworth and Mayer 1995). Because the selfing rates were estimated using seedlings, and there is evidence for differential survival of selfed zygotes from fertilization to the time of sampling of leaves for the electrophoretic scoring of seedling genotypes (see below), we corrected the estimates of selfing rates to take into account the loss of products of selfing (Stevens and Bougourd 1988; Karkkainen and Savolainen 1993). If the chance that a product of selfing survives to be scored is v times that of an outcrossed seed (v expected to be less than 1), then the zygote-stage selfing rate [S.sub.0] is given by

[S.sub.0] = [S.sub.1]/[S.sub.1] (1 - v) + v

where [S.sub.1] is the selfing-rate estimated using seedlings. The v values for the four populations can be found from the products of number of seeds per flower pollinated and germination rate. Table 3 shows these values, and the two selfing-rate estimates, using [S.sub.0] values from the multilocus estimation procedure. Given that the level of inbreeding depression was not very high under the conditions in which these seeds were generated and grown, it is not surprising that the correction only slightly affects the selfing-rate estimates, which range from 0.37-0.69.

Inbreeding Depression in the Four Populations

1. Results from the second-generation plants. - A moderate level of inbreeding depression, affecting many of the characters studied, was observed in the seeds and the greenhouse-grown second-generation plants (Table 4). Significant in-breeding depression was found for many of the characters measured (15 of the 40 tests, using two-tailed tests). Given the large number of tests performed, it is difficult to decide how to assess overall significance. It seems reasonable to evaluate the effects of inbreeding on each character separately, in which case a Bonferroni correction would give a P-value of 0.0125 as the criterion for the 5% level. In what follows, we have therefore taken tests significant at the 1% level as meaningful.

There were no significant effects of inbreeding up to the time of mature seed formation (no effects on fruits per flower pollinated, fruit maturation time, or number of seeds per fruit), in any population, except that in population T selfed flowers produced slightly fewer seeds than outcrossed flowers (P = 0.009). The pollination failure rate, as measured by number of fruits per flower pollinated and number of seeds per fruit, was not different in selfed versus outcrossed plants (except for seeds per fruit in population T), indicating a lack of early-acting inbreeding depression. Seeds of selfed flowers were highly significantly smaller in the D and H population experiment, even though there were more seeds in capsules derived from cross-pollinations, and seed weight was significantly negatively related to the numbers of seeds per capsule, for both self- and cross-pollinated flowers. There were no significant differences in seed weight with the smaller sets of results from plants from populations M or T. It is also worth noting here that there were no significant effects of pollination treatment on the mean weights of the seeds from any of the four populations that were planted to produce the plants whose fitness characteristics were studied, perhaps because of smaller sample sizes.

Seeds produced by selfing germinated more slowly than outcrossed seeds, although most seeds from both pollination treatments eventually germinated. The lack of germination in the early stages could have important consequences for competition among seedlings. Apart from early germination, there was no other character for which selfed seeds consistently performed worse than outcrossed progeny. Seedling mortality was somewhat higher for selfed seeds in population D, but not in H (this was not scored in the other two populations), and seedling growth over 10 wk was significantly lower in selfed progeny from populations D and M, although not in the other two populations. In population T, both the initial and final sizes of seedlings from selfing were slightly greater than those of seedlings derived from outcrossing, but these differences were not statistically significant.

Of the five characters studied on mature plants in each population (date of first flowering, final number of flowers on the first inflorescence, number of secondary branches, ovule numbers per flower and pollen stainability), selfed progeny performed worse than outcrossed progeny for plant height at 10 wk, date of first flowering, number of flowers on the first inflorescence, and number of secondary branches in populations D, H, and M, and number of secondary branches, number of ovules, and pollen stainability in population T (Table 4). However, these differences were small and most were not statistically significant. None of the five instances [TABULAR DATA FOR TABLE 4 OMITTED] in which the selfed progeny performed better than the outcrossed plants were significant.

In addition to the measurements of plant characters related to fertility just described, the fertility of plants from selfed and outcrossed seeds was assessed when seeds for the next-generation plants were being generated by self- and cross-pollinations (Table 5). Differences in fertility between the two cross types were slight. Inbred and outcrossed plants differed little in capsule formation per flower pollinated (fruit set in Table 5). Outcrossed plants from the D and H populations had much higher autogamy rates than selfed plants; these differences were significant at the 0.1% level by paired [TABULAR DATA FOR TABLE 5 OMITTED] t-tests comparing the values for self and outcross progeny of the same maternal plants within populations. Autogamy data were not collected for progeny from populations M and T.

Numbers of seeds and their size are less useful than the characters described above as measures of fertility, because differences might be caused by differences in the inbreeding coefficient of the seeds, rather than that of the maternal plant, whereas it seems reasonable to assume that the number of fruits per flower pollinated is chiefly a maternal plant characteristic. For the outcrossed hand pollinations, however, all progeny should be highly outbred and can thus distinguish the effects, if any, of maternal level of inbreeding. Although outbred maternal plants had higher seed numbers per fruit than inbred plants in three of the four populations (Table 5), only one comparison was significant (population M), whereas in population D there was a significant effect in the opposite direction. The results for seed weight were similar, with three out of the four comparisons showing greater seed weight for outbred parent plants with significant effects in the D and T populations, whereas the M population effect was significant in the opposite direction. Overall, therefore, the results suggest a small effect of inbreeding on female fertility.
TABLE 6. Selfing rates and overall relative fitness values obtained
by multiplying effects found at different stages of the life cycle.


                                          Population
                                D         H         M         T


Estimated selfing rate        0.52      0.37      0.69      0.49


Second-generation plants


Seed number per fruit         0.931     0.911     1.138     0.836
Germination rate              0.982     0.893     0.906     0.984
Survival rate                 0.963     0.965       -         -
Flower number (1st infl.)     0.837     0.897     0.847     1.016
Number of branches            0.915     0.919     0.943     0.906
Fruits per pollination        0.933     0.932     1.132     0.996


Total Product


Excl. survival                0.783     0.671     0.823     0.757
Incl. survival                0.755     0.647       -         -
Incl. fertility               0.704     0.603     0.932     0.754


Third-generation plants


Seed number per fruit         0.925     0.889     0.883     0.855
Germination rate              0.957     1.032     0.982     0.996
Survival rate                 1.076     0.952     0.912     0.903
Flower number (1st infl.)     1.082     1.006     0.895     0.874
Number of branches            0.960     0.928     0.964     0.895


Total product


Excl. survival                0.920     0.857     0.748     0.688
Incl. survival                0.989     0.816     0.682     0.622


The data from the different characters studied on the second-generation plants were combined into an overall measure of the estimated mean fitnesses of selfed progeny relative to outcrossed progeny by multiplying together fitness components that can be assumed independent; this yields a quantity equal to 1 minus the estimated inbreeding depression (Table 6). Inbreeding depression occurred at most life-cycle stages for the second-generation plants (for a discussion of the third-generation plants, see below), although it was usually slight. Overall inbreeding depression (total product) followed the expected relationship with the breeding system; that is, the M population had the highest selfing rate and the lowest inbreeding depression (highest relative fitness), whereas the most outcrossed population, H, had the highest inbreeding depression. The other two populations had selfing rates and inbreeding depression intermediate to M and H. The method based on inbreeding-depression estimates for sets of inbred and outcrossed progeny of individual maternal plants (suggested by Johnston and Schoen 1994) yielded high standard errors of the inbreeding-depression estimates (which is not unexpected because only two progeny plants per maternal parent were measured for most of the characters), and the differences between the effects of one generation of inbreeding between the populations are not statistically significant by this test.

2. Results from the Third-Generation Plants: Effect of the Inbreeding Coefficient on Fitness. - For the third-generation plants, 11 characters were studied in plants of each population for each of four inbreeding coefficients. Twenty-five of the 44 characters showed statistically significant (at the 1% level) inbreeding effects in the expected direction: lower values with increasing inbreeding coefficient or, for mean dates of seed germination and of flowering, increased values ([ILLUSTRATION FOR FIGURES 1-2 OMITTED], Table 7). Only three of the 44 slopes were not in the expected direction (germination and date of flowering in population M and ovule number in population T; [ILLUSTRATION FOR FIGURES 1-2 OMITTED]). Table 7 shows the linear-regression coefficients of the natural logarithm-transformed character values on the inbreeding coefficients, together with tests of their significance obtained by jackknife. Twenty-five of the 44 regressions were significant at the 1% level. As in the previous generation, there were no significant differences in seed weight between pollination treatments for the subset of seeds that were planted. It is therefore unlikely that seed weight differences are the cause of the differences found in the progeny plants.

Overall measures of the estimated mean relative fitnesses of selfed and outcrossed progeny were calculated the same way as for second-generation plants. For the third-generation plants, only the progeny from the XX and XS pollinations (see Table 2) were used, because these estimate the effects of a single generation of selfing. Inbreeding depression occurred at most life-cycle stages and was relatively slight, as with the second-generation plants (Table 6). When populations that were grown in the same block were compared (D versus H, and M versus T), again inbreeding depression decreased with an increase in the selfing rate.

Selfed and outcrossed plants in the third generation should differ more in inbreeding coefficient than do the second-generation plants (0 and 0.5 for the comparison of XX and XS plants in the third generation, compared with F (the inbreeding coefficient) and (1 + F)/2 for the comparison of X and S progeny in the second generation, or a difference of [TABULAR DATA FOR TABLE 7 OMITTED] [TABULAR DATA FOR TABLE 8 OMITTED] (1 - F)/2, which is less than 0.5 if F is greater than zero). One would therefore expect higher inbreeding depression to be expressed in the third generation. For two populations, M and T, there was, however, less inbreeding depression in the third-than in the second-generation progeny, although the opposite was true for the other two populations.

In addition to inbreeding depression within the four populations, we looked for evidence of heterosis in several characters when plants from different populations were crossed, that is, the hybrid progeny having values greater than either parent [ILLUSTRATION FOR FIGURE 1-2 OMITTED]. Only one case was found, involving flower numbers in the H x D crosses, but in other cases the hybrids were intermediate between the means of the parental strains, and there were no instances of outbreeding depression.

In the tests for multiplicative versus synergistic effects of inbreeding, no significant effects were found for the 11 characters tested in each of the four populations (by Spearman rank correlations between the residuals for progeny of different inbreeding coefficient). There is thus little sign of important deviations from the multiplicative model, although it is difficult to quantify the extent of nonlinearity that could have been present but was undetectable in our study.

3. Effect of Competitive Growing Conditions. - In the competitive conditions, selfing significantly reduced height in three of the four populations, compared with plants grown singly (Table 8). Mean height of plants grown under crowded conditions was reduced by 50% in three of the populations, compared with plants grown singly, but in the T population, crowded plants were 20% taller. The magnitude of the effect of inbreeding was, however, similar in three of the four populations. Comparing ratios of selfed to outcrossed height for maternal families that were grown in both conditions, the ratio was similar in crowded and singly grown plants from the H population (1% lower when crowded). In the T population, the difference between progeny of selfing and outcrossing was statistically significant only in the competitive conditions, and the ratio 18% greater (i.e., inbreeding depression was greater in competitive conditions). In the D and M population samples, the ratios were 6% and 3% lower, respectively, in competitive conditions, and the effect of selling on height was not statistically significant in either experiment in the D population. There is thus no evidence for a great difference in inbreeding depression in plant height, under conditions that had strong effects on height itself, except possibly in the T population. For plant height, the more outcrossed population in each pair (H and T) showed a greater height reduction in selfed versus outcrossed plants as compared to the selfed populations, but the more outcrossed populations had a greater dry weight ratio (S/X) than the selfed populations.

DISCUSSION

In agreement with the finding of fairly high levels of selfing in the field, plants from all populations produced seed autogamously in the greenhouse, with autogamy rates ranging from 0.26 for population D to 0.47 for population M (Charlesworth and Mayer 1995). The selfing rates were not well correlated with the autogamy rates, which is not surprising in view of the small range of selfing rates found for the four populations. The electrophoretically determined selfing rates yielded no values as low as those reported by Weil and Allard (1964), who based estimates on a flower color genetic marker, and reported that C. heterophylla is largely an outcrossing species.

The effects of one generation of inbreeding in plants from the populations studied were mild, with significant effects being detected mainly for characters measured at the early stages of the life cycle, although plant heights and, to some extent, fertility were also significantly reduced by inbreeding. In most of the tests done with plants of the four populations, self-pollination produced fewer mature seeds, with lower mean weights, and these seeds germinated more slowly, than seeds from outcrosses. Selfed seedlings suffered higher mortality and grew more slowly than seedlings from outcrosses. The selfed progeny plants flowered a few days later than their outcrossed half-sibs, and tended to have fewer branches and flowers, as well as slightly lower probabilities of fruit maturation per hand-outcrossed flower, although many of these differences were not individually significant. These differences were found in both the second- and third-generation seeds and plants. The largest magnitude of total average inbreeding depression was estimated at less than 40% (see Table 6).

The values calculated in Table 6 might be underestimates, because the plants were grown in individual pots and given regular fertilizer treatment, such that growth and vigor were probably much higher than in the field. Certainly flower numbers as high as those of the greenhouse-grown plants were not seen in the field. Inbreeding depression might be less severe under these conditions. It is well known that environmental conditions can affect the magnitude of inbreeding depression (e.g., Schemske 1983; Dudash 1990; Schmitt and Ehrhardt 1990; Karoly 1991; Johnston 1992; Wolfe 1993). Although plants grown in highly competitive conditions were stunted, they exhibited similar effects of inbreeding on plant height to those found in the better growth conditions; thus the magnitude of inbreeding depression for this character was not markedly greater under these conditions. Overall, it therefore appears that these populations have mild inbreeding depression, which is consistent with their being fairly highly inbreeding, as indicated by the selfing-rate estimates between 0.37 and 0.69.

To examine the relationship between selfing rate and inbreeding depression for individual fitness characters, we compared D versus H and M versus T because the populations were raised in pairs in blocks. Both comparisons between more- and less-inbred populations showed higher relative fitness of selfed progeny in the more-inbred population, in both generations of plants (Table 6). However, the differences are not great and are not easily subjected to tests of significance. The effects on individual characters of a single generation of inbreeding are slight, and estimates of the relative values for selfed progeny have large errors. Furthermore, the differences in the selfing-rate estimates between populations are not extreme. Even if one assumes that the populations have maintained these selfing rates for many years, one would expect that their genetic loads would differ only slightly. Given the fact that the selfing-rate estimates represent values for a single year in each population, and outcrossing might well vary from year to year (Willis 1993b), and given the errors of the inbreeding-depression estimates, one would therefore not expect close agreement.

The conclusion that the populations differ in genetic load is, however, somewhat supported by the fact that inbreeding depression declines with an increase in the selfing rate for both second- and third-generation plants. In comparisons between populations grown in the same blocks (D and H, M and T), examination of the regression slopes on the inbreeding coefficient for the individual characters studied (Table 7) shows significant differences between populations in the slopes in six of the 22 comparisons (for height at 1 wk, date of first flower, and ovule number in the data set from populations D and H, and for seed number per fruit, probability of germination, and survival to flower in the M and T population comparison). In every significant comparison, the population with the lower selfing rate showed the greater effect of inbreeding.

Tests of whether genetic loads decrease with the level of inbreeding should probably compare populations with large differences in outcrossing rates, and differences in inbreeding depression have indeed been found between populations differing widely in their selfing rates (see above). Other species of Collinsia may be more appropriate for such comparisons than C. heterophylla. For instance C. grandiflora vats. grandiflora and parviflora (originally considered separate species, C. grandiflora and C. parviflora) are distinguished on the basis of striking differences in flower size, and are reported to vary greatly in outcrossing rate (Ganders and Krause 1986). Collisia grandiflora var. parviflora is autogamous, whereas var. grandiflora sets little seed if pollinators are excluded. Unfortunately the flowers of var. parviflora are extremely small and difficult to emasculate without damage (Mayer and Charlesworth, unpubl. data), making cross-pollination impossible. However, outcrossing rates have not been estimated from other species of Collinsia other than C. verna; thus it remains possible that contrasting breeding systems that are easier to work with may be found in other species in the genus. For example, C. sparsiflora var. arvensis has also been reported to differ widely in breeding system between populations (Schemske and Lande 1985).

It is nevertheless clear that, despite their mixed mating systems and moderate levels of inbreeding, the populations of C. heterophylla we studied have substantial genetic load and exhibit inbreeding depression within populations. This is clearly evident when regressions of fitness characters on inbreeding coefficients are analyzed. Effects on all stages of the life cycle studied were found, including adult plant characters, such as flower number, that would contribute to fertility. In the absence of pollen discounting (Holsinger et al. 1984), a necessary condition for outcrossing to evolve is the existence of inbreeding depression exceeding 0.5 (Charlesworth et al. 1990), and changes in selfing rates are weakly selected when inbreeding depression is close to 0.5. The inbreeding-depression levels in the populations appear to be close to 40%, or even 50% (if plant size is taken as an estimate of fitness); thus, the intermediate selfing rates of these populations are not very surprising.

Our data suggest that there is no strongly synergistic effect of the different genetic factors that contribute to decline in fitness under inbreeding. Here the true inbreeding coefficient of the progeny from two generations of selfing must exceed 0.75 because the populations are partially inbreeding, implying that the seeds collected in the field must have nonzero average inbreeding coefficients. Assuming that the selfing rates estimated represent equilibrium values, the F-values of these seeds should be given by S/(2 - S) (Crow and Kimura 1970) and should range from 0.23 for population H to 0.53 for population M. This would yield F-values after two generations of selfing of 0.81 and 0.88 for these two populations, respectively, with the other populations having values between these extremes. The higher of these values is substantially greater than the expected value of 0.75 after two generations of selfing. This should lower the fitness of the progeny of two generations of selfing, relative to the fitnesses of the plants with the other F-values, and might produce apparent synergistic effects. Despite this, fitness expressed on a logarithmic scale declined close to linearly with inbreeding coefficient; that is, no such effects were detected, suggesting that there is no strong acceleration in fitness decline, even at high inbreeding levels. Because the slopes are slight, however, fitness decline on a log scale would be only slightly greater than linear; thus a slight degree of synergism could not be detected. Our conclusions agree with those of Willis's (1993a) from outcrossing natural populations of Mimulus guttatus, those of McCall et al. (1994) on Impatiens capensis, and with results from crop plants (Hallauer and Sears 1973; Wright 1977), where linear fitness decline with increased inbreeding coefficient was found.

ACKNOWLEDGMENTS

We thank B. C. Husband and D. W. Schemske for permission to cite their unpublished work, and D. Campbell and two anonymous reviewers for comments on the manuscript. We also thank the greenhouse staff, S. Yamins, S. Suwanski, and J. Zdenek, for plant care.

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Author:Mayer, Stephanie S.; Charlesworth, Deborah; Meyers, Blake
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Date:Apr 1, 1996
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