Heredity 86 (2001) 184±194 Received 16 November 1999, accepted 17 October 2000

Inbreeding depression in two populations of Arenaria uni¯ora (Caryophyllaceae) with contrasting mating systems

LILA FISHMAN Department of Ecology and Evolutionary Biology, Princeton University, Princeton, NJ 08544 U.S.A.

I used parallel family-structured crossing designs to investigate the relative performance of self and outcross progeny in sel®ng and predominantly outcrossing populations of the annual plant Arenaria uni¯ora. The selfer population experienced much lower inbreeding depression (d ˆ 0.05 ‹ 0.02 SE) than the outcrossers (d ˆ 0.19 ‹ 0.02 SE). The negative association between genetic load and sel®ng rate suggests that purgable partially recessive alleles are the primary source of inbreeding depression, as does its late expression in both populations. Inbreeding depression in the selfer population, which naturally consists of highly inbred lines, was used to calculate the mean dominance (h ˆ 0.33) and incidence rate (U ˆ 0.30) of deleterious mutations. In the outcrosser population, signi®cant variation among individuals in the expression of inbreeding depression may re¯ect lineage-speci®c di€erences in inbreeding history or, more probably, random variation in mutational load. The low (0.5) inbreeding depression of outcrossers suggests that the maintenance of a mixed mating system in some A. uni¯ora populations and the evolution of nearly cleistogamous self-pollination in others may re¯ect local pollinator-mediated selection for sel®ng rather than the constant 3:2 genetic advantage invoked by many models.

Keywords: Arenaria uni¯ora, genetic load, inbreeding depression, mating system, sel®ng.

Introduction contrast, inbreeding depression due to nearly additive mildly deleterious alleles or overdominance at ®tness Inbreeding depression is the major force opposing the loci can persist in inbred populations (Charlesworth & evolution of self-fertilization in ¯owering plants and is a Charlesworth, 1990). However, in a population with a central element in most theories of mating system long history of inbreeding, variation at overdominant evolution (e.g. Lande & Schemske, 1985; Lloyd, 1992). loci should be lost through drift or selection for the The mating system and genetic load of a population more ®t homozygote, and inbreeding depression due to interact. If the expression of deleterious partially reces- overdominance also reduced. Empirical studies of the sive alleles is the primary source of inbreeding depres- relationship between inbreeding depression and sel®ng sion, the equilibrium genetic load will re¯ect the rate in natural populations generally ®nd decreases in population's past history of inbreeding (Lande & genetic load with increased sel®ng rate, consistent with Schemske, 1985; Charlesworth & Charlesworth, 1987, the purging of deleterious alleles. (Carr & Dudash, 1996; 1990; Charlesworth et al., 1990b). High sel®ng rates Husband & Schemske, 1996; Johnston & Schoen, 1996). facilitate ecient `purging' of such alleles by exposing However, the relationship between inbreeding depres- them to selection in the homozygous state, and feedback sion and sel®ng rate is often only weakly negative (Latta between reductions in genetic load and outcrossing rate & Ritland, 1994), and many highly sel®ng populations will lead to the evolution of complete sel®ng once maintain substantial levels of inbreeding depression inbreeding depression drops below a threshold value set (Holtsford & Ellstrand, 1990; AÊ gren & Schemske, 1993; by selection for self-fertilization (Lande & Schemske, Charlesworth et al., 1994). 31985; Charlesworth et al., 1990b; Lande et al., 1994). In Variation in inbreeding depression among individu- als, as well as the population mean, may be important in Correspondence and present address: Department of Biology, Duke mating system evolution. The development of associa- University, Box 90338, Durham, NC 27708-0338 U.S.A. E-mail: tions between loci a€ecting ®tness and those controlling 2 l®[email protected] mating system can allow high sel®ng rates to evolve even

184 Ó 2001 The Genetics Society of Great Britain. INBREEDING DEPRESSION IN ARENARIA 185 when deleterious recessives cause population mean populations used in this study represent the extremes of inbreeding depression greater than selection for sel®ng mating system within the species. The outcrosser popu- (Holsinger, 1988; Uyenoyama & Waller, 1991). This lation (Pendergrass, GA; Wyatt's [1984] population 7), results in the joint divergence of mating behaviour and is highly protandrous and individual outcrossing rates genetic load among families, and has focused attention measured in naturally pollinated arrays indicate a mixed on individual levels of inbreeding depression rather than mating system (t ˆ 0.77 ‹ 0.09 SE; Fishman, 2000). In the population mean (Uyenoyama et al., 1993). contrast, plants from the selfer population (Liberty, SC; Depending on the genetic basis of inbreeding depres- Wyatt's [1984] population 8) are nearly cleistogamous, sion, such associations may also allow the maintenance generally self-pollinating in the bud. Low rates of pollen of mixed mating. import from outcrossers (<10%) and export to out- Measures of inbreeding depression in natural popu- crossers (<2%) indicate the likelihood of pollen transfer lations provide a quantitative context for understanding among selfers is vanishingly low (Fishman, 2000). ecological and genetic selection for self-fertilization. Despite their striking ¯oral and mating system diver- Here, I examine inbreeding depression in two popula- gence, the Liberty, SC and Pendergrass, GA populations tions of the annual plant Arenaria uni¯ora (Caryo- are geographically and genetically allied. The popula- phyllaceae) with contrasting mating systems. These tions occur on small granite outcrops separated by populations have been well characterized in previous 100 km and both populations consist of large numbers studies of A. uni¯ora and represent extremes of sel®ng of individuals with mean densities at ¯owering of at least rate and ¯oral morphology within the species (Wyatt, 500 plants/m2 (Wyatt, 1986). Allozyme and crossability 1984, 1986; Fishman & Wyatt, 1999; Fishman, 2000). data indicate that the Liberty selfer population is more The evolution of autonomous sel®ng in A. uni¯ora closely related to the Pendergrass outcrossers than to appears be a case of reproductive character displace- more distant A. uni¯ora populations, including other ment resulting from local pollinator-mediated competi- selfers (Wyatt, 1990; Wyatt et al., 1992). See Wyatt tion (Fishman & Wyatt, 1999). This provides a rare (1984, 1986) for detailed locations and descriptions of opportunity to examine inbreeding depression in a these populations. species in which the reproductive assurance value of sel®ng has been quanti®ed. Crossing design In this paper, I present estimates of the timing and magnitude of reductions in inbred progeny performance Parallel family structured crossing designs were used to in selfer and outcrosser A. uni¯ora and discuss factors examine the ®tness consequences of sel®ng in the two a€ecting genetic load in the two populations. To further populations. This approach allows estimation of within- explore the genetic basis of inbreeding depression in population variation in genetic load and the construc- A. uni¯ora, I calculate the dominance and mutation rate tion of con®dence intervals around population mean of deleterious alleles in the selfer population using inbreeding depression (Johnston & Schoen, 1994). methods developed for highly inbred lines (Charles- Within each population, the performance of self and worth et al., 1990a). I examine the distribution of outcross half-sib families was measured across life stages inbreeding depression among families within each from seed to reproductive adult. population and consider the role of within-population Dormant rosettes of selfers and outcrossers were variation in genetic load in mating system evolution in collected in early March 1996 and transplanted into A. uni¯ora. Finally, I compare the magnitude of 8 cm square plastic pots containing a thin layer of ®eld inbreeding depression in these populations to the soil over coarse sand. The plants were raised in an ecologically contingent bene®ts of self-fertilization. insect-free greenhouse at the University of Georgia, which was kept cool (15°C) and naturally lit throughout the growing season. Before ¯owering, plants from each Materials and methods population were randomly selected as recipients for self and outcross hand-pollinations (N ˆ 32 selfers and 36 Study populations outcrossers). The crossing design was slightly di€erent Arenaria uni¯ora (Walt.) Muhl. (Caryophyllaceae) is a for the two populations because of the pseudocleisto- diploid (2n ˆ 14) winter annual plant endemic to granite gamy of the Liberty selfer population. outcrops in the south-eastern United States. The species Flowers of outcrossers were emasculated at anthesis consists of large-¯owered protandrous populations and hand-pollinated 3±4 days later when the stigmatic (outcrossers) in the centre of its range and small- lobes became receptive. In addition to outcross and ¯owered autonomously sel®ng plants (selfers) in margi- self treatments for estimating inbreeding depression, the nal populations (Wyatt, 1984, 1986). The two A. uni¯ora Pendergrass plants received ®ve other pollination

Ó The Genetics Society of Great Britain, Heredity, 86, 184±194. 186 L. FISHMAN treatments discussed elsewhere (Fishman & Wyatt, 1999). vernalization, then warmed to 15±20°C to initiate shoot Outcrossed ¯owers received pollen from ³2 donors per production and ¯owering. Daylength was not manipu- pollination from a separate pool of 100 donor plants. lated, but mercury vapour lamps provided supplemental Selfed ¯owers received pollen from younger ¯owers on light during the day. The end of the ¯owering season the maternal plant. About ®ve ¯owers on each plant was e€ected by allowing the plants to gradually dry out. received each treatment over a 4-week period, with an Progeny arrays from the two populations were grown on e€ort made to pollinate each plant with both treatments adjacent benches, rather than intermixed, because dif- on any given day. A lack of fruit set by emasculated ferences in size between selfers and outcrosser plants control ¯owers indicated that there was little illicit would have resulted in asymmetric competition between pollen transfer in the insect-free greenhouse. the populations, a potentially greater confounding Because Liberty selfer plants routinely self-pollinate factor. in the bud, it was necessary to emasculate selfer ¯owers Some families from each population had complete several days prior to anthesis. To reduce work and germination failure in one or both pollination treat- wastage of damaged ¯owers, only outcross treatment ments and were excluded from postgermination tests of ¯owers were emasculated. Under a dissecting micro- performance to balance the design. Thirty-two outcros- scope, the petals and anthers were removed with forceps ser families and 26 selfer families provided sucient and the stigma inspected for pollen. The stigma and seedlings for inclusion in the progeny arrays. Progeny ovary were inspected again after 2±3 days and, if free of were grown in square trays consisting of 64 25 cm2 cells, pollen and undamaged, pollinated with outcross pollen. so that each replicate block contained a full complement Outcross pollination of selfers consisted of mixed pollen of self and outcross families (32 dams ´ 2 cross types). from ³ 2 donors from a separate pool of selfer plants. For the selfers, families with excess seedlings were Self ¯owers were allowed to set seed autonomously substituted for missing families, so that six maternal because preliminary crosses had indicated that supple- families (both self and outcross) were replicated within mental hand-pollination does not increase the seed set of blocks. Families were assigned random positions within selfers. Approximately four ¯owers on each plant each array. received outcross pollen and six ¯owers were allowed Because intraspeci®c competition or other stresses can to set seed autonomously. Because A. uni¯ora ¯owers increase inbreeding depression (e.g. Schemske, 1983; generally close and begin to form fruits within six hours Dudash, 1990; Wolfe, 1993), I measured postgermina- of pollination (Fishman, personal observation), it is tion performance at two densities. Half of the total unlikely that ¯owers receiving outcross pollen had number of arrays (outcrosser N ˆ 10; selfer N ˆ 6) were autonomously self-pollinated. planted at high density (six seedlings per cell) and half at low density (two seedlings per cell). Crowding in the low density arrays was comparable to late season ®eld Measurement of inbreeding depression densities of both populations. Seedlings that died within Mature fruiting capsules from all crosses were collected two weeks of transplanting (<5%) were replaced with 2±3 weeks after pollination. At the end of the season, seedlings from the same cross. This mortality did not the seeds from each treatment ´ maternal plant combi- vary across populations or other variables and was nation were pooled, then counted and weighed in bulk. probably due to damage during transplanting. Subse- Seed set (seeds per ¯ower) and mean seed mass (mg) quent mortality was very low (<2%). Total progeny were calculated. Germination of each seed pool was performance was measured for each cell and divided by assayed in Para®lm-sealed Petri dishes containing moist the original planting density. Thus, mortality is included sand. Seeds were after-ripened at 30°C/15°C (12 h/12 h in the performance measures, as is compensatory growth cycle) in an environmental control chamber for three by siblings within the cell. months, then cooled to 15°C/6°C to initiate germina- I obtained three measures of adult performance: shoot tion. Total percentage germination was recorded after production, early ¯owering and fecundity. Shoot num- one month of cool temperatures. ber was counted when plants were still in the rosette Post-germination components of ®tness were meas- stage, before shoot elongation and ¯owering. An early ured in a greenhouse at Princeton University. Although count of ¯owers was made eight weeks after transplant- it was not possible to test progeny performance in the ing, when some families had not yet begun to ¯ower, ®eld, growing conditions were chosen to mimic natural and estimates the timing of ¯owering. Fecundity was conditions. As with the parental generation, plants were measured at the end of the ¯owering season by counting grown in a thin layer of ®eld soil over coarse sand and all ¯owers and fruits. The persistent sepals and pedicels watered from below until well-established. The green- of A. uni¯ora allow counting of past ¯owers even when house was kept at 10°C for six weeks to ensure no fruits have formed. In the nearly cleistogamous

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selfers, ¯ower number is a good estimator of female depression (d ˆ 1 ) ws/wo, where ws and wo are the ®tness because most ¯owers have full fruit and seed set ®tness of selfed and outcrossed progeny, respectively) in the absence of pollinators (Fishman, 1998). Outcros- for each maternal family. Because there was no evidence ser fruit set is more dependent on the pollination of inbreeding depression of seed characters, mean self environment, so ¯ower production puts an upper bound and outcross fecundities (the back-transformed LSMs of on female fecundity. the dam ´ cross type interaction from the ANOVA) were used to calculate lifetime inbreeding depression. How- ever, analyses using a composite measure of ®tness (seed Statistical analyses set ´ percent germination ´ fecundity) produced similar Analyses were performed using the standard least mean values of inbreeding depression. squares analysis of variance platform in JMP 3.0.2 Population-level inbreeding depression was calculated (SAS Institute, 1994). Progeny performance after sel®ng as the mean of family d-values. The mean of ratios does and outcrossing was analysed separately for the Liberty not necessarily equal the ratio of means, so this family and Pendergrass populations. Maternal identity (dam) structured estimate of inbreeding depression is not was included as a random e€ect in all ANOVA models. identical to the population-level measure used in most Cross type (selfed vs. outcrossed) was the main ®xed models of mating system evolution (Johnston & Schoen, e€ect. Analyses of particular response variables deviated 1994). However, the relatively large number of adult from the general model as described below. progeny measured in each dam ´ cross type family

Seed performance variables were analysed with the (nselfers ˆ 6 cells or 24 plants, noutcrossers ˆ 10 cells or 40 general ANOVA model, which is equivalent to a paired plants) assures that the mean of family level inbreeding t-test. Seed set and seed mass were analysed directly. depression in this study is close to the population mean. Germination rates were arcsine±square root transformed In simulations, Johnston & Schoen (1994) found that before analysis to normalize the residuals. Because seeds family sizes >5 produce unbiased estimates of popula- from each dam ´ cross type combination were pooled for tion-level inbreeding depression. The conventional met- measurement, these analyses cannot identify between- ric of inbreeding depression (d ˆ 1 ) ws/wo) generates family di€erences in response to sel®ng. unbounded negative values when ws exceeds wo (i.e. Adult performance variables were analysed with a when individual families show outbreeding depression), more complex model, which included competition but does not bias the population mean at the range of treatment and replicate blocks nested within competi- relative ®tnesses observed in this experiment. A sym- tion treatment as well as family and cross e€ects. All metrical estimator of inbreeding and outbreeding two-way interaction terms involving cross were included depression (relative performance [RP]; AÊ gren & Schem- in the ®nal ANOVA model, but other two-way and three- ske, 1993) produced values nearly identical to those way interactions that were nonsigni®cant (P > 0.2) in a reported. preliminary full analysis were pooled with the error term. For F-tests, JMP 3.0.2 (SAS Institute, 1994) Results generates synthetic denominators based on interaction terms and the residual. For this model, all main e€ects Fitness consequences of sel®ng across life stages other than cross were tested over the e€ect ´ cross interaction and all interactions were tested over the Neither population experienced inbreeding depression residual. Cross e€ects were tested over a synthetic of seed performance (Fig. 1). Within the Pendergrass denominator MS. The adult performance measures were outcrossers, there were no signi®cant di€erences ln-transformed and the data conformed to ANOVA between self and outcross pollinations in the number assumptions after transformation. This transformation of seeds produced per fruit, mean seed mass or makes the signi®cance of two-way interactions an proportion germinated. The Liberty selfer population appropriate test of di€erences between families or appeared to experience signi®cant outbreeding depres- environments in inbreeding depression (Kalisz, 1989; sion of seed set (Fig. 1). This apparent outbreeding Johnston & Schoen, 1994). In the outcrossers, a depression in the Liberty population probably resulted constant (0.25) was added to the shoot and ¯ower from the emasculation of only outcross treatment values prior to transformation in order to include zero ¯owers, which sometimes damaged the recipient ovary. values. Overall inbreeding depression within each life Visibly damaged ¯owers were not used in the experi- stage was indicated by signi®cant di€erences in per- mental crosses, but some ovary damage may have gone formance between selfed and outcrossed progeny. undetected and reduced seed set in this treatment. The To examine the distribution of genetic load within di€erent prepollination treatment of selfed and out- and between populations, I calculated inbreeding crossed selfer ¯owers is unlikely to contribute to

Ó The Genetics Society of Great Britain, Heredity, 86, 184±194. 188 L. FISHMAN

patterns of ®tness at later life stages because it did not signi®cantly a€ect other measures of seed performance (Fig. 1) and the seed characters were never signi®cant covariates in analyses of adult fecundity (data not shown). Both populations exhibited strong family e€ects on components of seed performance across pollination treatments. The outcrossers showed highly signi®cant maternal e€ects on both seed weight and percentage germination (P < 0.0001). A negative correlation between seed number and seed weight (Pearson corre- lation; r ˆ )0.37, P < 0.0001) and a positive correlation between seed mass and germination rate (r ˆ 0.50, P < 0.0001) suggest shared genetic or maternal e€ects. In the selfers, maternal identity strongly in¯uenced seed set and seed mass (P < 0.0001), but did not a€ect germination rate. As in the outcrossers, seeds per fruit and seed mass were negatively correlated (r ˆ )0.32, P ˆ 0.01). After seedling establishment, the magnitude of inbreeding depression in each population changed across the growing season (Fig. 2). In both selfers and outcrosser populations, outcrossed progeny produced 20% more shoots than selfed progeny and cross type was a highly signi®cant e€ect in the ANOVA. In the outcrossers, both shoot number and inbreeding depres- sion of shoot production were dependent on maternal identity (P < 0.01). In the selfers, shoot number did not depend on maternal identity (P ˆ 0.21), but levels of inbreeding depression of this character varied widely among families (P < 0.001). Signi®cant inbreeding depression of early ¯ower production occurred in the selfers, but not in the outcrossers (Fig. 2). Families in both populations varied signi®cantly in early ¯ower production, but the e€ect of maternal identity was much stronger in the outcrossers (P < 0.0001 vs. P ˆ 0.018), because many outcrosser plants were not yet ¯owering. In both populations, families di€ered substantially in inbreeding depression of early ¯owering (P < 0.0001). At the end of the season, levels of inbreeding depression in the two populations were again quite di€erent, with the outcrossers exhibiting greater inbreeding depression (Fig. 2; Table 1). The e€ect of cross was highly signi®cant in the outcrossers

(F1;18.4 ˆ 68, P < 0.0001), with d ˆ 0.19 ‹ 0.02 SE calculated across families. Cross type also signi®cantly Fig. 1 Seed character means (‹1 SE) after self and outcross a€ected fecundity in the selfers (F1;5.6 ˆ 8.4, P ˆ 0.03) hand-pollinations of plants from outcrosser and selfer popu- but overall inbreeding depression was lower lations of Arenaria uni¯ora. Means are cross type e€ect LSMs (d ˆ 0.05 ‹ 0.02 SE calculated across families). The (back-transformed if necessary) from separate analyses of variance for each population. Inbreeding depression (or, in the fecundity of individuals in both populations was highly case of selfer seed set, apparent outbreeding depression) is correlated with early shoot number (Pearson correlation indicated by signi®cant di€erences between self and outcross of LSMs; rselfers ˆ 0.72 and routcrossers ˆ 0.68). This performance (***P < 0.001). suggests that di€erences in the initial production of

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¯owering shoots may determine later patterns of repro- duction. Maternal identity had an only marginally signi®cant e€ect on fecundity across pollination treat- ments (P ˆ 0.022 and 0.064, respectively, for selfers and outcrossers). With the exception of early ¯owering and shoot production in the selfer population, density had a strongly signi®cant e€ect on adult performance (P < 0.005). Replicate arrays also varied signi®cantly in mean performance for all characters (P < 0.005). In contrast, neither competition treatment nor array a€ec- ted the relative performance of self and outcross progeny at any life stage, as indicated by the consistent nonsigni®cance of cross ´ array and cross ´ density interaction e€ects in both populations (P > 0. 20).

Correlation of relative self and outcross performance across adult stages Although the magnitude of inbreeding depression changed substantially across the growing season, the ranking of families remained fairly constant. In selfers, family level inbreeding depression of total fecundity was positively correlated with relative performance at both the shoot production (r ˆ 0.63; P < 0.001) and early ¯owering stages (r ˆ 0.43, P < 0.05). The outcrossers showed similar across-family correlations between inbreeding depression at the end of the season and at earlier stages. Although no population-level inbreeding depression of early ¯owering was detected in the outcrossers, variation among families in d of fecundity was correlated with relative performance at early ¯owering (r ˆ 0.51, P < 0.005).

Distribution of inbreeding depression among families Selfers and outcrossers displayed di€erent amounts of among-family variation in inbreeding depression of fecundity (Table 1, Fig. 3). In the Pendergrass outcros- sers, a highly signi®cant dam ´ cross type interaction re¯ects consistent di€erences in inbreeding depression

among families (F31;558 ˆ 2.7871; P < 0.001). In con- trast, selfer families did not signi®cantly di€er from one another in the relative fecundity of selfed and outcrossed

progeny (F25,318 ˆ 1.15, P > 0.25). In the Pendergrass outcrossers, more than two-thirds of the families exhib- Fig. 2 Adult performance means (‹1 SE) of self and outcross ited signi®cant inbreeding depression. In the selfer progeny from outcrosser and selfer populations of Arenaria population, only six of 26 families experienced signi®cant uni¯ora. Means are back-transformed cross type e€ect LSMs inbreeding depression and eight families exhibited non- (which average across families, densities and arrays) from signi®cant outbreeding depression (negative d-values). separate analyses of variance for each population. Inbreeding Although family mean values of d cover a wide range in depression is indicated by signi®cant di€erences between self both populations, variation in performance within selfer and outcross performance (***P < 0.001, *P < 0.05). families overshadows the among-family variation.

Ó The Genetics Society of Great Britain, Heredity, 86, 184±194. 190 L. FISHMAN

Table 1 Nested mixed-model analyses Population Source d.f. MS FPof variance for the fecundity of self Outcrosser Cross type 1 8.11 68.41 0.000 and outcross progeny of Arenaria (Pendergrass, Dam 31 0.25 1.74 0.064 uni¯ora. The outcrosser and selfer GA) Density 1 171.47 926.38 0.000 populations were analysed separately Array[density] 8 0.19 7.25 0.006 with parallel experimental designs and Cross ´ dam 31 0.15 2.79 0.000 ANOVA models. Cross type (self vs. Cross ´ density 1 0.03 0.83 0.388 outcross) is a ®xed e€ect and dam is a Cross ´ array[density] 8 0.03 0.49 0.863 random maternal family e€ect. Array Error 558 0.05 is a random block e€ect nested within the ®xed e€ect of planting density. Selfer Cross type 1 0.31 8.42 0.029 Fecundity values (total fruit and/or (Liberty, Dam 25 0.10 2.28 0.022 ¯ower production) were ln-trans- SC) Density 1 55.21 33.19 0.005 formed for analysis. The same ANOVA Array[density] 4 1.66 54.78 0.001 model was used to analyse the other Cross ´ dam 25 0.04 1.15 0.285 measures of adult performance Cross ´ density 1 0.00 0.02 0.893 Cross ´ array[density] 4 0.03 0.80 0.526 Error 318 0.04

depression of ®tness. Inbreeding depression of total fecundity was much higher in the Pendergrass outcrosser population (mean d ˆ 0.19 ‹ 0.02 SE) than in the Liberty selfers (mean d ˆ 0.05 ‹ 0.02 SE). Estimates of population-level inbreeding depression made without explicitly incorporating family structure were 0.22 and 0.06, respectively, for outcrossers and selfers.

Discussion

The magnitude and timing of inbreeding depression The lack of inbreeding depression of seed performance in both A. uni¯ora populations is consistent with the purging of early acting lethal recessive alleles. Even moderate levels of inbreeding (through sel®ng or during recent population bottlenecks) should be sucient to remove such alleles from a population (Lande & Schemske, 1985; Charlesworth & Charlesworth, 1987). Fig. 3 Distribution of inbreeding depression of fecundity (d)in Other empirical studies have also con®rmed this predic- the outcrosser and selfer populations of Arenaria uno¯ora. tion (Husband & Schemske, 1996). Each bar is the mean d for a single maternal family (N ˆ 32 Lower inbreeding depression of fecundity in the and 26 dams for outcrossers and selfers, respectively). The obligate selfers relative to the outcrossers also supports hatched bars indicate families with inbreeding depression the theory that genetic load due to deleterious recessives signi®cantly di€erent from zero in the ANOVA. The horizontal evolves jointly with sel®ng rate (Lande & Schemske, lines show the mean (solid) and 95% con®dence interval 1985; Charlesworth & Charlesworth, 1987, 1990; (dotted) of inbreeding depression for each population as a Charlesworth et al., 1990b). However, in a population whole. with a long history of routine inbreeding, ®xation of the higher-®tness allele (given any asymmetry of homozy- gote ®tness) would also eliminate genetic load due to overdominance at ®tness loci Charlesworth & Charles- Population-level effects of inbreeding worth, 1990). In addition, drift may ®x deleterious On average, plants from outcrosser and selfer popula- alleles in highly sel®ng populations. Without actually tions experienced di€erent levels of inbreeding reducing the genetic load, such ®xation could reduce

Ó The Genetics Society of Great Britain, Heredity, 86, 184±194. INBREEDING DEPRESSION IN ARENARIA 191 measured inbreeding depression (the lower ®tness of Low levels of inbreeding depression could also result selfed relative to outcrossed individuals), because all if the genetic load was not fully expressed under these plants carry the same alleles. Very low inbreeding experimental conditions. Perhaps inbreeding depression depression in highly sel®ng populations (e.g. this study, would have been higher under more stressful ®eld Barrett & Charlesworth, 1991; Parker et al., 1995; conditions, which can amplify ®tness asymmetries. Husband & Schemske, 1996; Johnston & Schoen, 1996) Some comparative studies have found increased is probably due to a combination of these processes. inbreeding depression in the ®eld (Schemske, 1983; Both A. uni¯ora populations exhibit total inbreeding Dudash, 1990) or under competitive regimes (Schmitt & depression at the low end of the spectra for their 5Ehrhardt, 1990; Wolfe, 1993), whereas others have observed mating systems. The values found in this study detected no di€erence in self and outcross relative are much lower than the mean cumulative inbreeding performance between environments (Charlesworth depression values for sel®ng (d ˆ 0.22) and outcrossing et al., 1994; Nason & Ellstrand, 1995). In this study, (d ˆ 0.49) ¯owering plants reviewed by Husband & higher density (3´) clearly provided a stressful environ- Schemske (1996). The moderate late-acting inbreeding ment, dramatically reducing ®tness in both populations depression in the Pendergrass outcrosser population (outcrossers F1;8 ˆ 926.4, P < 0.001; selfers F1,4 ˆ 33.2, (sel®ng rate <0.3, d ˆ 0.19) resembles that of a typical P < 0.001). However, increased competition had no sel®ng species. The Liberty selfers, which have a sel®ng detectable e€ect on the expression of inbreeding depres- rate close to 1.0, experience negligible inbreeding sion in either population. Although measuring total depression of total fecundity (d ˆ 0.05). Similarly low performance for each cell rather than for single individ- inbreeding depression has been reported for other highly uals could have allowed vigorous self progeny to sel®ng populations, including Jamaican Eichornia pan- partially compensate for cellmates homozygous for iculata (Barrett & Charlesworth, 1991) and two species deleterious alleles, such compensation would also have of Amsinckia (Johnston & Schoen, 1996). Other studies reduced the overall e€ect of competition on ®tness. have found substantial inbreeding depression even in Because the strong, highly signi®cant environmental highly sel®ng taxa (Holtsford & Ellstrand, 1990; Karoly, e€ects on performance (density and array) did not a€ect 1991; Lyons & Antonovics, 1991; AÊ gren & Schemske, the expression of genetic load, it is also highly unlikely 1993). Low (<0.20) levels of inbreeding depression in that the observed between-population di€erence in intermediately outcrossing species are less frequently inbreeding depression results from any environmental reported, although some mixed-mating Collinsia popu- di€erence between the benches. lations appear similar to the A. uni¯ora outcrossers (Mayer et al., 1996). Estimation of dominance and mutation A number of factors (other than mating system per se) rate from inbreeding depression in selfers which reduce e€ective population size may contribute to the relatively low levels of inbreeding depression The extremely high sel®ng rate of the Liberty selfer observed in these Arenaria uni¯ora populations. Both population provides the opportunity to make additional morphs of A. uni¯ora are restricted to isolated and highly inferences about the genetic load in this species. If one seasonal granite outcrops, and drastic ¯uctuations in assumes multiplicative e€ects of ®tness loci and muta- population size may have been a signi®cant part of their tion±selection balance, inbreeding depression due to population genetic histories. Biparental inbreeding and deleterious alleles in a completely self-fertilizing popu- sel®ng after colonization events or other extreme demo- lation is determined only by the dominance coecient graphic bottlenecks could have substantially reduced the (h) and the mutation rate (Charlesworth et al., 1990a). genetic load of the currently mixed mating Pendergrass The harmonic mean degree of dominance can be population (Lande & Schemske, 1985). Although esti- calculated as the coecient of the regression of outcross mates of the population outcrossing rate based on adult ®tness (wij) on self ®tness (wii), where i indicates a heterozygosity at a single locus suggest that the Pender- maternal parent and j is a sire chosen from the grass population is not highly inbred (Fishman, 1998), population at random, a modi®cation the method of such estimates must be treated with caution because Johnston & Schoen (1995). Because maternal e€ects diversity at allozyme loci is low in all A. uni¯ora may also contribute to the regression of outcross on self populations (Wyatt et al., 1992). Measures of inbreeding sibling (parental) ®tness, some caution must be used in depression in additional selfer and outcrosser popula- interpreting estimated dominance coecients. In this tions of A. uni¯ora would help di€erentiate the roles of case, wij and wii were the cross ´ dam e€ect LSMs from demographic history and mating system in in¯uencing the ANOVA of fecundity. In Arenaria, the regression of inbreeding depression, although these can never be outcross on self sibling performance indicates partial entirely separated in natural populations. recessivity of deleterious mutations (h ˆ 0.33 ‹ 0.20 SE)

Ó The Genetics Society of Great Britain, Heredity, 86, 184±194. 192 L. FISHMAN

a€ecting fecundity in the selfer population. With only 26 that increase the outcrossing rate become associated families, this dominance coecient cannot be statisti- with high viability genotypes (Uyenoyama et al., 1993). cally discriminated from either complete additivity Di€erential purging, as well as such genetic associations, (h ˆ 0.5) or complete recessivity (h ˆ 0) of deleterious could create systematic variation in inbreeding depres- alleles. However, the value for A. uni¯ora is quite similar sion within mixed mating populations. However, the to estimates of dominance coecients from selfer individual variation in inbreeding depression observed populations of Amsinckia (Johnston & Schoen, 1995) in this and other mixed mating populations (e.g. Parker and inbred lines of Drosophila (Charlesworth & Charles- et al., 1995; Dudash et al., 1997) probably does not worth, 1987). re¯ect individual inbreeding history, because it may not The mutation rate (U) is approximated by U ˆ be possible to di€erentiate variation due to inbreeding 6[)2ln(1 ) d)]/1 ) 2h (Charlesworth et al., 1990a). With history from random variation in mutational load. a dominance estimate of 0.33 and mean inbreeding Random mutational variation can swamp variation depression of fecundity of 0.05, the estimated per due to inbreeding history, and variation at additive loci genome mutation rate in A. uni¯ora selfers is 0.30. This further prevents the detection of associations between mutation rate is similar to those calculated for loci individual genetic load and inbreeding depression a€ecting overall ®tness in two highly sel®ng populations (Schultz & Willis, 1995). Within-population variation of Amsinckia spectabilis (Johnston & Schoen, 1995), but in mutational load could provide an opportunity for the lower than estimates from two autogamous Leavenwor- establishment of a sel®ng modi®er that fortuitously thia populations with substantial levels of inbreeding occurs in a lineage with low inbreeding depression, but depression (Charlesworth et al., 1994). Although they this does not appear to have happened in the mixed do not have great statistical power, these calculations of mating A. uni¯ora population. No relationship was dominance and mutation rate are in accordance with the observed between individual autogamy rate and growing consensus that inbreeding depression in plants inbreeding depression in A. uni¯ora outcrossers (Pear- has a common source in partially recessive deleterious son correlation, r ˆ 0.17, P ˆ 0.63). Lineage-speci®c mutations (Charlesworth et al., 1990a). associations between mating behaviour and genetic load cannot be implicated in mating system evolution within this species. Variation in inbreeding depression among individuals Inbreeding depression relative to selection The two A. uni¯ora populations di€er not only in their for self-fertilization mean genetic load, but in the distribution of inbreeding depression among individuals. Maternal plants from the Inbreeding depression in the closely related Pendergrass mixed mating Pendergrass population vary widely in the population may be used to infer selection against self- relative ®tness of their self and outcross progeny pollination in outcrossing ancestors of the Liberty (Table 1; Fig. 3). In contrast, Liberty selfer maternal selfers. If sel®ng can be achieved without loss of plants do not signi®cantly di€er from one another in outcross male ®tness (i.e. if no `pollen discounting' their levels of inbreeding depression of fecundity 7occurs: Holsinger, 1988), inbreeding depression must be (Table 1, Fig. 3). Higher variability of inbreeding greater than 0.5 to counteract the 3:2 advantage of selfer depression in outcrossers has also been reported for a genes (Fisher, 1941; Jain, 1976). Thus, the population- pair of Epilobium species with contrasting mating level d observed in the Pendergrass outcrossers systems (Parker et al., 1995). This pattern may simply (0.19 ‹ 0.02 SE) is much lower than the threshold arise from the long history of inbreeding in the selfer value of 0.5 predicted by most models of the evolution population, in which each lineage continuously purges of sel®ng (Lloyd, 1979; Lande & Schemske, 1985; all but the most mild (or least recessive) new deleterious Charlesworth et al., 1990b). Under the assumptions of mutations. Homogenization of the selfer population by such models, the evolution of autogamy in the Liberty recent genetic bottlenecks could also contribute to population requires no further explanation and the reductions in both inbreeding depression and variation maintenance of predominant outcrossing in the Pen- in genetic load. dergrass population is dicult to explain. However, Variation among lineages in inbreeding depression, as additional processes appear to have been important in observed in the outcrosser population, been suggested as generating the current pattern of mating system vari- a major factor in mating system evolution (Holsinger, ation in A. uni¯ora. 1988; Uyenoyama & Waller, 1991; Uyenoyama et al., Localized pollinator-mediated selection appears to 1993). In particular, stable mixed-mating systems can have favoured the evolution of autonomous self-pollin- evolve when inbreeding depression is low and modi®ers ation in A. uni¯ora (Fishman & Wyatt, 1999). Selfers

Ó The Genetics Society of Great Britain, Heredity, 86, 184±194. INBREEDING DEPRESSION IN ARENARIA 193 only occur in areas of sympatry with congener A. glabra, based only on measures of inbreeding depression. whereas outcrossers and A. glabra never co-occur However, these results also emphasize that the magni- (Wyatt, 1984, 1986). In ®eld experiments, outcrossers tude of inbreeding depression alone cannot explain the su€ered a 20±35% reduction in female reproductive evolution of sel®ng or the maintenance of outcrossing, ®tness in the presence of A. glabra (Fishman & Wyatt, and supports theoretical approaches that consider 1999). These losses primarily result from heterospeci®c inbreeding depression in conjunction with empirical pollen transfer by shared pollinators, ovule usurpation, estimates of ecological and genetic selection for self- and the production of inviable hybrid seeds. Even in the fertilization (e.g. Lloyd, 1979, 1992; Holsinger, 1992; absence of an automatic transmission advantage, locali- Harder & Wilson, 1998). zed selection for pre-emptive self-pollination of this magnitude could have exceeded the moderate inbreeding Acknowledgements depression observed in current outcrossers. Experimen- tal measures of inbreeding depression and pollinator- I am very grateful to Robert Wyatt for sharing his mediated selection cannot fully reproduce ancestral extensive knowledge of Arenaria and for providing conditions, but these results do suggest a quantitatively logistical support for the research conducted in Georgia. important role for reproductive assurance in mating Thanks to Don Stratton, Hope Hollocher, John Willis, system evolution. Daniel Schoen, Chris Eckert and two anonymous Although the maintenance of predominant outcross- reviewers for helpful comments on earlier versions of ing in the Pendergrass population may simply re¯ect this manuscript. This research was supported by grants nonequilibrium conditions, it could also indicate con- from Sigma Xi and by NSF grant DEB-9623350 to straints on mating system variation. If sel®ng variants D. Stratton and the author. experience trade-o€s in male ®tness, such that increases in self-pollination reduce pollen export, no constant References selection for self-pollination occurs (Nagylaki, 1976; Holsinger, 1988) and sel®ng may not be favoured even AAGRENÊ GREN, J.AND SCHEMSKE , D. W. 1993. Outcrossing rate and when inbreeding depression is very low. In A. uni¯ora, inbreeding depression in two annual monoecious herbs, the morphological and phenological changes associated Begonia hirsuta and B. semiovata. Evolution, 47, 125±135. with autonomous self-pollination prevent outcross pol- BARRETT, S. C. H.AND CHARLESWORTH , D. 1991. E€ects of a len donation by the Liberty selfers, producing complete change in the level of inbreeding on the genetic load. Nature, 352, 522±524. pollen discounting (Fishman, 2000). These trade-o€s CARR, D. E.AND DUDASH , M. R. 1996. Inbreeding depression in could be secondary adaptations to the sel®ng syndrome. two species of Mimulus (Scrophulariaceae) with contrasting However, quantitative genetic analysis suggest that mating systems. Am. J. Bot., 83, 586±593. pleiotropy contributes to associations between protan- CHARLESWORTH, D.AND CHARLESWORTH , B. 1987. Inbreeding dry and ¯ower size in A. uni¯ora (Fishman, 1998). By depression and its evolutionary consequences. Ann. Rev. imposing trade-o€s between autogamous self-fertiliza- Ecol. Syst., 18, 237±268. tion and pollinator attraction, such a genetic architec- CHARLESWORTH, D.AND CHARLESWORTH , B. 1990. Inbreeding ture could also constrain the male ®tness of new sel®ng depression with heterozygote advantage and its e€ect on variants. Unfortunately, the magnitude and genetic selection for modi®ers changing the outcrossing rate. basis of pollen discounting in initial sel®ng variants Evolution, 44, 870±888. remains largely unknown, complicating predictions of CHARLESWORTH, B. D., CHARLESWORTHAND MORGAN , M. T. 8 1990a. Genetic loads and estimates of mutation rates in the levels of inbreeding depression required to maintain highly inbred plant populations. Nature, 347, 380±382. outcrossing. CHARLESWORTH, D., MORGAN, M. T.AND CHARLESWORTH , B. The joint evolution of inbreeding depression and 9 1990b. Inbreeding depression, genetic load, and the evolu- sel®ng rate, suggested here for Arenaria uni¯ora, can be tion of outcrossing rates in a multilocus system with no major factors in mating system evolution (Lande & linkage. Evolution, 44, 1469±1489. Schemske, 1985; Charlesworth & Charlesworth, 1987). CHARLESWORTH, D., LYONS, E. E.AND LITCHFIELD , L. B. 1994. 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Ó The Genetics Society of Great Britain, Heredity, 86, 184±194.