Ecol Res (2008) 23: 995–1003 DOI 10.1007/s11284-008-0467-3

O R I GI N A L A R T IC L E

Kiyoshi Ishida

Effects of inbreeding on the magnitude of inbreeding depression in a highly self-fertilizing tree, Magnolia obovata

Received: 15 June 2007 / Accepted: 13 January 2008 / Published online: 19 February 2008
The Ecological Society of Japan 2008

Abstract Inbreeding depression is one of the major selective forces driving the evolution of mating systems. Previous theories predict that long-lived plants will show a negative correlation between inbreeding depression and the level of inbreeding (as determined by an inbreeding coefficient) at maturity, but the extent of this correlation may vary among life stages because of variation in the genetic basis for inbreeding depression at different stages. To test this prediction, I used electrophoretic allozyme analysis and pollination experiments to examine the fixation index (Fis) at maturity and inbreeding depression in the early and late life stages of two populations with different outcrossing rates of a highly self-fertilizing tree, Magnolia obovata. The magnitude of inbreeding depression for early survival (de) in an outcrossing population (tm = 0.51; Fis =
0.015) was higher (de = 0.97) than that in an inbreeding population (tm = 0.18; Fis = 0.15; de = 0.38). From these results, I estimated that both populations exhibited high inbreeding depression for late survival (dl) (0.94 in the outcrossing population and 0.93 in the inbreeding one) and lifetime survival (dt) (0.99 and 0.96, respectively). My results and previously published data demonstrate the predicted relationship between inbreeding depression and the level of inbreeding for early survival, but not for late survival. This suggests that there is a differential genetic basis for inbreeding depression at different life stages. The inbreeding depression for late survival appears to play a central role in the maintenance of reproductive traits that promote outcrossing in M. obovata. Keywords Inbreeding depression Æ Inbreeding Æ Life stage Æ Purging of inbred load Æ Long-lived plants K. Ishida Kansai Research Center, Forestry and Forest Products Research Institute, Nagai-Kyutaro, Momoyama, Fushimi, Kyoto 612-0855, Japan E-mail: ishidak@affrc.go.jp Tel.: +81-75-6111201 Fax: +81-75-6111207

Introduction Inbreeding depression, which represents a reduction in the fitness of inbred offspring compared with that of outcrossed offspring, is a major selective force that drives the evolution of a plant’s mating system (Barrett and Harder 1996; de Jong and Klinkhamer 2005; Goodwillie et al. 2005). Theory predicts that successive generations of a population experiencing inbreeding will purge recessive deleterious mutations as a result of selection, resulting in a decreased magnitude of inbreeding depression (Lande and Schemske 1985; Charlesworth and Charlesworth 1987; Charlesworth et al. 1990). Here, I refer to the decrease that occurs in the magnitude of inbreeding depression as a result of inbreeding as ‘‘purging’’ of the inbred load. The theory also predicts that the extent of this purging depends on the genetic basis of the inbreeding depression: that is, inbreeding depression due to lethal mutations can be purged more easily than inbreeding depression due to mildly deleterious mutations. Prediction of the purging of the inbred load forms the basis of some theories of the evolution of mating systems in plants (Lande and Schemske 1985; Porcher and Lande 2005) and has led to empirical studies of the relationship between inbreeding and inbreeding depression (Husband and Schemske 1996; Byers and Waller 1999). Previous studies have argued that long-lived plants exhibit a high genomic mutation rate per generation because of recessive mitotic mutations in apical meristems between the zygote stage and the mature plant (Klekowski and Godfrey 1989; Klekowski 1998), and this high genomic mutation rate is thought to cause more severe inbreeding depression than is the case in short-lived plants (Williams and Savolainen 1996; Lynch and Walsh 1998; Morgan 2001). As the extent of purging of the inbred load depends on the secondary selfing rate (i.e., the proportion of selfed progeny remaining after selection resulting from inbreeding depression) under a high genomic mutation rate per generation (Lande et al.

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1994), the magnitude of inbreeding depression in longlived plants may be negatively correlated with the level of inbreeding at maturity (as determined by the secondary selfing rate or an inbreeding coefficient). Furthermore, this relationship between inbreeding depression and the level of inbreeding may vary among life stages, since the genetic basis of inbreeding depression is thought to vary among life stages (Husband and Schemske 1996; Goodwillie and Knight 2006). Because trees generally require a large number of cell divisions before flowering, and this may result in a high genomic mutation rate per generation (Klekowski 1988; Petit and Hampe 2006), studies of inbreeding depression in tree populations will provide an opportunity to test the theoretical predictions for long-lived plants. Few studies have examined the relationship between inbreeding depression and the level of inbreeding in tree species. Previous studies have suggested that purging of the inbred load occurs at the embryonic stage in some tree species (Ka¨rkka¨inen et al. 1996; Hirayama et al. 2007), but no studies have examined variation in the extent of purging among life stages in natural tree populations. An understanding of the relationship between life-history strategy and the maintenance of inbreeding depression thus requires intraspecific studies of inbreeding depression at each life stage of tree populations with different levels of inbreeding at maturity. Measuring the magnitude of inbreeding depression expressed by natural tree populations is generally difficult because of the long lifespans of trees. However, if the outcrossing rates at the seed stage and the inbreeding coefficients at maturity can be measured, then the magnitude of the inbreeding depression in survival between seed and maturity can be estimated indirectly from these two parameters (Ritland 1990). This indirect method will provide better estimates than direct estimation based on cultivation experiments, because the conditions in a greenhouse or nursery (with a benign environment) often cause lower inbreeding depression than is observed under field conditions, where the environment generally imposes more stress on the plants (Hauser and Loeschcke 1996; Cheptou et al. 2000), resulting in the underestimation of inbreeding depression in cultivation experiments. Here, I examined the purging of the inbred load in a tall tree, Magnolia obovata (syn. M. hypoleuca), which reproduces by mixed mating with a high frequency of self-fertilization (Ishida et al. 2003; Isagi et al. 2004). Recently, I suggested that this species might exhibit substantial inbreeding depression despite its high frequency of self-fertilization (Ishida 2006). Moreover, a preliminary electrophoretic study of M. obovata found among-population variation in outcrossing rates (Ishida and Nakamura 1997; K. Ishida, Kansai Research Center, unpublished data). The species thus provides an opportunity for studying the purging of the inbred load in tree species. To estimate the effects of inbreeding on inbreeding depression in M. obovata, I selected two populations

with different outcrossing rates (as determined by a preliminary electrophoretic study; unpublished data) and then conducted allozyme analysis and pollination and germination experiments with these populations to measure the magnitude of inbreeding depression for four traits: early survival (from zygote to germinating seed), seed mass, late survival (from seedling to maturity), and lifetime survival (from zygote to maturity). On the basis of these results and previously published data on another population of the species (Ishida et al. 2003; Ishida 2006), I discuss the extent of purging in each component of inbreeding depression and its evolutionary implications for the species.

Materials and methods Study species Magnolia obovata is a common tall tree that reaches heights of up to 20 m and is found in temperate forests in Japan. The flower is protogynous and hermaphroditic, with 100–150 carpels. Individual carpels always have two ovules. The flower’s protogyny prevents autogamy, but allows geitonogamous selfing because of asynchronous flowering of individual flowers on the same tree (Ishida et al. 2003). Geitonogamous selfing reduces the seed set, and a previous study demonstrated that this reduction is due to inbreeding depression rather than pre-zygotic self-incompatibility (Ishida et al. 2003). According to a previous microsatellite analysis of seedlings (Isagi et al. 2000), the pollen flow distance from donor to recipient flowers is 3–540 m (average 131 m). Birds favor the seed coat (outer portion of the outer seed coat) and thus disperse the seed over long distances. Study populations I obtained preliminary estimates of outcrossing rates at the seed stage in three populations in northern Japan (Misumai, Jozankei, and Hitsujigaoka, near Sapporo, in Hokkaido Prefecture) and two in southern Japan (northern and southern areas of Aburayama, near Fukuoka, in Fukuoka Prefecture) to select study populations for use in this study; 124–273 seeds were collected from 8 to 10 trees in each population between 1995 and 1998 and were analyzed for 3 or 4 allozyme loci (as described in the ‘‘Electrophoretic analysis’’). The outcrossing rates obtained in this preliminary analysis ranged from 0.10 to 0.66 [K. Ishida, Kansai Research Center, unpublished data; data for the Hitsujigaoka population were reported by Ishida et al. (2003)]. Of the five populations, the two with the highest and lowest outcrossing rates (Misumai and the northern Aburayama population, respectively) were selected for this study. The study site of the Misumai population was located in central Hokkaido (4255¢N, 14116¢E, elevation 450 m asl) and that of the Aburayama population was in

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northern Kyushu (3331¢N, 13022¢E, elevation 300 m asl). The Misumai trees grew in a deciduous broadleaved forest dominated by Acer mono, Tilia japonica, and Quercus crispula, and the Aburayama trees grew in a mixed forest dominated by Quercus serrata, Castanopsis cuspidata, and Pinus densiflora. The two populations are large, and each appears to include more than 1,000 trees. Each study area was ca. 5 ha. The density of mature trees (those with a diameter at breast height, DBH, greater than 4 cm) at Misumai was 11.6 trees per ha, and that at Aburayama was 12.2 trees per ha. Electrophoretic analysis Outcrossing rates at the seed stage were estimated by using 124–273 seeds per population per year (average number of seeds = 221); the seeds were collected from seven to ten trees in 1996 (Misumai), 1998 (Aburayama), and 1999 (both populations). Population genetic parameters (heterozygosity and fixation index) at maturity were estimated in winter buds from 60 randomly chosen trees (DBH > 4 cm) in each population. Seeds and bud materials were stored at 5C before electrophoretic analysis. For electrophoresis, the seeds (excluding the seed coat) and buds (ca. 100 mg of leaf primordia) were ground in 200 and 10,00 ll, respectively, of cold extraction buffer (Tsumura et al. 1990). Electrophoresis was performed with vertical-slab polyacrylamide gels at 4C, a current of 12.3 mA/cm2, and a duration of 150 min. The following six enzymes were analyzed: aspartate aminotransferase (AAT; EC 2.6.1.1), alcohol dehydrogenase (ADH; EC 1.1.1.1), diaphorase (DIA; EC 1.6.*.*), leucine amino-peptidase (LAP; EC 3.4.11.1), menadione reductase (MNR; EC 1.6.99.2), and 6-phosphogluconate dehydrogenase (6PGD; EC 1.1.1.44). Genetic interpretations of the resulting banding patterns were performed on the basis of their segregation patterns with respect to typical subunit structures (Richardson et al. 1986; Kephart 1990). Seven loci were then used as population genetic parameters at maturity (Aat-1, Aat-2, Adh, Dia, Lap, Mnr, 6Pgd). In the analysis of the seeds, because of their poor activity Adh, Mnr, and 6Pgd were not scored, and Dia could not be scored in the Misumai population because of poor resolution. Although the seed materials contained not only embryos but also endosperm, which is a triploid tissue with two maternal genes and one paternal gene per locus, I could estimate genotypes of the embryos at the remaining three polymorphic loci [Aat-1, Aat-2, and Lap for Misumai and Aat-1, Aat-2, and Dia (excluding Lap for reasons given below) for Aburayama], since microsatellite analysis of M. obovata seeds on the basis of five loci [M6D3, M6D4, M10D3, M10D8, and M17D5 (Isagi et al. 1999)] suggested that the maternal genes of an endosperm are identical to the maternal gene of the embryo in the same seed (Ishida K, Kansai Research Center, unpublished data). The abovementioned sets of three polymorphic

loci were used to estimate outcrossing rates in the two populations. Lap was not used for Aburayama, since this locus was monomorphic in this population. To evaluate outcrossing rates at the seed stage, the multilocus (tm) and single-locus (ts) outcrossing rates based on the three loci were estimated by using version 3.0 of the MLTR program (Ritland 2002a, b). The difference between the two values (tm
ts) indicates the magnitude of biparental inbreeding. The method of Manly (1997) was used to calculate the 95% bootstrap confidence intervals for the outcrossing rates from bootstrap samples obtained by 1,000 resamplings of families. The observed and expected heterozygosities at maturity, based on the seven loci and their standard errors, were calculated, and the difference in the expected heterozygosity between the two populations was tested by the method of Nei (1987). The fixation index at maturity (Fis; Wright 1969) was calculated with version 2.9.3 of the FSTAT program (Goudet 1995, 2001). This value is equivalent to the inbreeding coefficient unless there is spatial genetic structure within a population (Nei 1987). The Fis values were tested for deviation from the Hardy–Weinberg equilibrium by using a 5,000-example randomization. Pollination and germination experiments I conducted pollination and germination experiments to measure the magnitude of inbreeding depression for early survival. Early survival was divided into two components: germination rate and embryo survival (from zygote to seed before germination). In the pollination experiment, manual self- and cross-pollinations were performed from May to June 2000 on five maternal trees per population to measure the embryo survival rate. Eight to ten recipient flowers from each maternal tree were used for self-pollination, and the same number of recipient flowers from each maternal tree plus one pollen donor tree located more than 50 m from the maternal trees was used for cross-pollination. Manual self- and cross-pollinations were conducted on flower buds 1 day before anthesis, after removal of the tepals and anthers. To perform the manual pollinations, the stigmas were brushed with pollen-producing anthers. The flower buds were covered by nylon netting after manual pollination to prevent natural pollination, and the netting was removed after the end of anthesis. Fruits from the manually pollinated flowers were collected from September to October, and the numbers of carpels and seeds in each fruit were counted. The embryo survival rate (me) of selfed and crossed progeny of each maternal tree was then calculated as me = [(number of fruits)/(number of flowers)] · (mean seed set ratio per tree), where the seed set ratio = (number of seeds per fruit)/(number of initial ovules per fruit). In addition, the fresh seed mass (the mass of seed with a developed seed coat) of every seed was weighed.

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The germination rates of selfed and crossed seeds were measured from 2001 to 2003 by using 51–360 seeds per treatment per maternal tree (average number of seeds = 235), as follows. The seeds were stored over the winter on moist filter paper at 5C to promote synchronous germination. In early May 2001, the seeds were placed on moist filter paper in Petri dishes in an incubator (12 h day/night; 25C day/5C night) in accordance with the method of Katsuta (1998). The seeds were allowed to germinate from late May to early July. Because seeds of the same cohort can germinate over a 2-year period in this species (Satoo 1992), ungerminated seeds were stored again at 5C in early August, and their germination was repeated again in the next year. The germination rates of selfed and outcrossed seeds of each maternal tree were calculated as: Germination rate = (number of seeds that germinated over 3 years)/(initial number of seeds).

population) indicates that the magnitude of the inbreeding depression differs among maternal trees. ANOVA was performed with SPSS version 14.0 software (SPSS 2005). In addition, to clarify whether not only recessive deleterious mutations but also post-zygotic self-incompatibility (uniform failure of self-fertilized ovules due to late-acting self-incompatibility before the cellular stage of endosperm development; Seavey and Bawa 1986) can be responsible for inbreeding depression and its between-population difference, I analyzed effects of selfing on the proportion of seeds aborted before endosperm development [(number of aborted ovules per fruit)/(number of initial ovules per fruit)] for manually pollinated fruits from three of the five maternal trees per population; this proportion corresponds to that of aborted ovules and/or seeds smaller than 2 mm in mature fruits (Ishida et al. 2003). I used ANOVA to analyze the effects as in the analysis for the embryo survival rate.

Inbreeding depression for early survival and seed mass Magnitude of inbreeding depression was evaluated by using the d value, where d = 1
(mean fitness of selfed progeny per population)/(mean fitness of crossed progeny per population) (Johnston and Shoen 1994). The values for embryo survival rate, germination rate, and seed mass were denoted as ds, dg, and dm, respectively. The jackknife standard error for these values was calculated by using the maternal tree in accordance with the method of Manly (1997). Cumulative inbreeding depression for early survival (de) was then calculated as de = 1
(1
ds)(1
dg). To examine the statistical significance of inbreeding depression (i.e., the difference in fitness components between selfed and crossed progeny) and its inter-population and inter-tree variations, I used mixed-model ANOVA to analyze the embryo survival rate, germination rate, and seed mass for selfed and crossed progeny. In these analyses, I examined the effects of crossing type and population (Misumai vs. Aburayama) as fixed factors and maternal tree (nested by population) as a random factor. The interaction between crossing type and population was also examined. The interaction between crossing type and maternal tree (nested by population) was examined only for seed mass, since replicates could not be obtained from each maternal tree for the two other traits. The embryo survival rate and germination rate were arcsine-transformed before the analyses to meet the assumptions of normality of residuals in the ANOVA. In these analyses, if the effect of crossing type is statistically significant and if the d value is positive, then we can conclude that significant inbreeding depression occurs in selfed progeny for both populations. If the interaction between crossing type and population is significant, then we can say that the magnitude of inbreeding depression differs between the populations. In addition, a significant interaction between crossing type and maternal tree (nested by

Inbreeding depression for late and lifetime survival I indirectly estimated inbreeding depression for late survival (dl) and that for lifetime survival (dt). To obtain these two values, I indirectly estimated the magnitude of inbreeding depression for survival from seed (just after the seed had completed development) to maturity (di) for the populations by using the method of Ritland (1990): di = 1
(2tm Fis )/[(1
tm )(1
Fis )],

ð1Þ

where Fis represents the fixation index at maturity. This method estimates the magnitude of inbreeding depression by evaluating the extent of the decrease in the inbreeding coefficient due to selection between the seed stage and maturity on the basis of the tm and Fis values at marker loci. Thus, F*, which denotes the fixation index at the same loci used for calculating the tm value (Aat-1, Aat-2, and Lap for Misumai; Aat-1, Aat-2, and Dia for Aburayama), was used instead of Fis in Eq. (1) to estimate di. In the estimation of di, two estimates and their average were calculated for each population, since two tm values (for 2 years) were used for the estimation in each population. The standard error (Se) of the mean di was then calculated for each population as se = (se1 + se2)/4, where se1 and se2 are the standard errors of di for 1 year (1996 for Misumai or 1998 for Aburayama) and the other year (1999 for both populations), respectively. The se1 and se2 values were calculated by using 1,000 bootstrap iterations for the maternal trees. In addition, I calculated di on the basis of two loci common to the two populations to clarify whether the between-population difference in the loci used affected the di based on the three loci. In these calculations, I assumed that the inbreeding coefficient at maturity equaled zero (i.e., di = 1.0) when the F* value was negative.

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The magnitude of inbreeding depression (dl) for late survival was estimated by using dg and di as follows: dl = 1
(1
di )/(1
dg ).

ð2Þ

As in the calculation of di, two estimates for dl and their mean were calculated for each population. Finally, cumulative inbreeding depression (dt) for lifetime survival was calculated using ds and di as dt = 1
(1
ds) (1
di).

Results Level of inbreeding and genetic diversity There was a substantial difference in the level of inbreeding between Misumai and Aburayama (Table 1). The Fis value at maturity, based on the seven allozyme loci for Misumai, was significantly higher than zero (P = 0.002), whereas that for Aburayama was not different from zero (P = 0.588), indicating that the former was an inbreeding population and the latter was an outcrossing population. The Fis value for Misumai was 0.15, which is approximately equivalent to that of progeny from a half-sib crossing (i.e., Fis for the first generation from a half-sib crossing = 0.125). The outcrossing rates at the seed stage, based on the three loci (tm and ts) for the two populations, were less than 1.0 (i.e., the upper limit of the 95% confidence interval for both values was lower than 1.0; Table 2), indicating that trees in both populations produced at least some seeds by means of self-fertilization. Both outcrossing rates for Misumai were substantially lower Table 1 Number of alleles per locus (na), mean observed heterozygosity (Ho), expected heterozygosity (He), and fixation index at maturity (Fis), based on seven allozyme loci in Magnolia obovata Population

na

Ho

He

Misumai

2.29

Aburayama

1.86

0.276 (0.068) 0.291 (0.089)

0.325 (0.041) 0.287 (0.045)

Fis 0.15
0.015

Standard errors appear in parentheses

than those for Aburayama; mean tm and ts values (average over 2 years) for the Misumai population were less than half the values for the Aburayama population. The differences between the tm and ts values were small in both populations (ranging from
0.04 to 0.03), indicating that the level of biparental inbreeding was low in both populations. Between-year variation in these values was substantial in both populations: for example, the differences in tm values between years were larger than half the mean tm value in both populations. The expected heterozygosity did not differ significantly between the two populations (Table 1; P > 0.05). The number of alleles per locus was larger at Misumai than at Aburayama. Inbreeding depression for early survival There was a substantial difference between the two populations in the magnitude of inbreeding depression for embryo survival rate (ds); the ds value for Misumai was less than half the value for Aburayama (Table 3). ANOVA for embryo survival rate showed that the effect of crossing type and the interaction between crossing type and population were both significant (Table 4), indicating a significant difference in the magnitude of inbreeding depression for embryo survival rate between the populations. The effect of population on embryo survival rate was also significant, but that of maternal tree (nested by population) was not. The proportion of seeds aborted before endosperm development for selfed fruits was larger than that for outcrossed fruits (for Misumai and Aburayama, respectively, mean ± SD, n = 12 and n = 11: for selfed fruits, 0.38 ± 0.18 and 0.43 ± 0.23; for outcrossed fruits, 0.08 ± 0.10 and 0.11 ± 0.14). The effect of crossing type on this value was significant (F = 7.97, df = 1, P = 0.048), indicating that post-zygotic selfincompatibility could contribute to inbreeding depression. The effect of interaction between crossing type and population was not significant (F = 0.02, df = 1, P = 0.895). Although selfing reduced embryo survival rates, selfed seeds were heavier than crossed seeds in both populations (i.e., dm < 0; Table 3). ANOVA for seed

Table 2 Multilocus (tm) and mean single-locus (ts) outcrossing rates and their difference (tm
ts), based on three allozyme loci in Magnolia obovata Population

Yeara

No. of samplesb

tm

Misumai

1996 1999 Mean 1998 1999 Mean

124 (8) 275 (9)

0.10 0.26 0.18 0.66 0.34 0.51

Aburayama

273 (10) 211 (7)

tm
ts

ts (0.04–0.17) (0.19–0.34) (0.56–0.78) (0.24–0.43)

The 95% confidence intervals appear in parentheses a Year when seeds were sampled b Number of seeds; number of maternal trees appears in parentheses

0.07 0.22 0.20 0.67 0.37 0.49

(0.02–0.12) (0.16–0.28) (0.56–0.79) (0.27–0.47)

0.03 0.03 0.03
0.01
0.04
0.02

(0.01 to 0.05) (0.02 to 0.06) (
0.04 to 0.03) (
0.061 to
0.008)

1000 Table 3 Mean values (± standard error) of embryo survival rate, mean seed mass, and germination rate, and magnitude of inbreeding depression (d value ± jackknife standard error) in two Magnolia obovata populations in relation to crossing type (selfed vs. outcrossed)

Embryo survival rate (%) Mean seed mass (g) Germination rate (%)

Misumai

Aburayama

Mean ± SE

Mean ± SE

Selfed

Outcrossed

d

Selfed

Outcrossed

d

27.9 ± 5.1 (5) 39.5 ± 4.7 (5) 21.0 ± 7.3 (5)

41.0 ± 6.2 (5) 33.7 ± 4.1 (5) 21.3 ± 7.0 (5)

0.32 ± 0.20
0.17 ± 0.13 0.02 ± 0.28

4.8 ± 3.1 (5) 30.5 ± 3.2 (5) 5.4 ± 3.3 (4)

37.9 ± 5.6 (5) 24.5 ± 2.4 (5) 25.6 ± 10.3 (5)

0.87 ± 0.07
0.24 ± 0.22 0.79 ± 0.10

Number of maternal trees appears in parentheses

Table 4 Results of ANOVA for embryo survival rate, seed mass, and germination rate in relation to crossing type (selfed vs. outcrossed), population, and maternal tree in the two Magnolia obovata populations

Traits

Source of variation

df

MS

F

P

Embryo survival rate

Crossing type Population Maternal tree (population) Crossing type · population Crossing type Population Maternal tree (population) Crossing type · population Crossing type · maternal tree (population)

1 1 8 1 1 1 8 1 8

0.517 0.215 0.027 0.158 5367.0 12979.7 27500.4 0.2 4949.7

27.23 8.07 1.40 8.32 6.72 3.01 5.56 0.00 147.05

<0.001 0.022 0.322 0.020 0.030 0.120 0.013 0.987 <0.001

Crossing type Population Maternal tree (population) Crossing type · population

1 1 8 1

0.042 0.025 0.055 0.069

3.29 0.47 4.30 5.39

0.112 0.513 0.035 0.053

Seed mass

Germination rate

mass demonstrated that the effect of crossing type was significant, whereas interaction between crossing type and population was not, indicating that the magnitude of inbreeding depression for seed mass was similar in the two populations (Table 4). The effects of maternal tree (nested by population) and of the interaction between crossing type and maternal tree were also significant. There was a large difference in inbreeding depression for germination (dg) between the populations; the dg value for Misumai was nearly zero, whereas that for Aburayama was ca. 0.8 (Table 3). In the ANOVA for germination rate, the effect of the interaction between crossing type and population was significant (Table 4), indicating different inbreeding depression for germination between the populations. The effect of maternal tree (nested by population) was also significant, but that of population was not significant. The magnitudes of the cumulative inbreeding depression for early survival (de) at Misumai and Aburayama were 0.38 and 0.97, respectively, indicating that the magnitude of inbreeding depression for early survival at Misumai was less than half the value at Aburayama.

Inbreeding depression for late and lifetime survival Both populations exhibited a high level of inbreeding depression (di) for survival from seed to maturity: di

values based on the three loci for Misumai and Aburayama were 0.94 ± 0.01 and 0.99 ± 0.00 (estimate ± se), respectively. The F* values for Misumai and Aburayama, which were used for the estimation of di, were 0.111 and 0.006, respectively. In addition, di values based on the two loci common to Misumai and Aburayama were similar to those based on the three loci: 0.96 ± 0.01 (F* = 0.098) and 1.0 (F* =
0.058), respectively, indicating that the effect of the betweenpopulation difference in the loci used was small for the di values based on the three loci. The populations also exhibited severe inbreeding depression for late survival (dl) from seedling to maturity; dl values for Misumai and Aburayama were 0.94 ± 0.01 and 0.93 ± 0.06, respectively. The magnitudes of the cumulative inbreeding depression for lifetime survival (dt) at Misumai and Aburayama were 0.96 and 0.99, respectively.

Discussion Level of inbreeding The allozyme analysis for the fixation index demonstrated that, with respect to gene transmission to the next generation, Misumai was an inbreeding population, whereas Aburayama was an outcrossing population. In a mixed mating population without spatial structure, the level of inbreeding is expected to be determined by both

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the outcrossing rate at the zygote or seed stage and by inbreeding depression expressed after this stage; hence, the large difference in the outcrossing rate between the two populations appears to have contributed partly to the difference in inbreeding level. The difference in outcrossing rates might reflect regional variation in pollinator activity, since M. obovata shows substantial regional variation in pollinator composition; the predominant pollinators in northern Japan (Hokkaido) are beetles (especially small beetles; Oedemeridae, Scraptiidae, Nitidulidae) (Kikuzawa and Mizui 1990; K. Ishida, Kansai Research Center, unpublished data), whereas those in southern Japan (central Honshu and Kyushu) include beetles, syrphids, bumblebees, and other bees (Yasukawa et al. 1992; K. Ishida, Kansai Research Center, unpublished data). In M. obovata populations, not only geitonogamy, but also biparental inbreeding would affect the level of inbreeding. However, the difference in the level of inbreeding between the two populations cannot be explained by biparental inbreeding, because their levels of biparental inbreeding were both low. Inbreeding depression for early survival and seed mass The pollination and germination experiments revealed that the outcrossing population (Aburayama) exhibited more severe inbreeding depression for early survival than the inbreeding population (Misumai). Although post-zygotic self-incompatibility could contribute to the inbreeding depression, it cannot be responsible for the between-population difference in inbreeding depression, since the effect of interaction between crossing type and population was insignificant for the proportion of seeds aborted before endosperm development. In previous studies (Ishida et al. 2003; Ishida 2006), another M. obovata population (denoted Hitsujigaoka) in northern Japan that had an outcrossing rate (tm based on four loci; Aat-1, Aat-2, Dia, and Lap) and fixation index (Fis based on six loci; Aat-1, Aat-2, Adh, Dia, Lap, and 6Pgd) that were intermediate (tm = 0.41; Fis = 0.08) between those of the Misumai and Aburayama populations also exhibited intermediate inbreeding depression for early survival: ds = 0.76 ± 0.08 and dg = 0.38 ± 0.14 (estimates at the population level ± jackknife standard error). Accordingly, both my results and previous ones suggest that M. obovata populations exhibit a negative correlation between the magnitude of inbreeding depression for early survival and the level of inbreeding at maturity. This trend supports the prediction of previous theories that the magnitude of inbreeding depression decreases as the level of inbreeding increases in a population of long-lived plants. In plant populations, genetic drift would contribute indirectly to a negative correlation between the magnitude of inbreeding depression and the level of inbreeding at maturity, since theory predicts that genetic drift decreases the magnitude of inbreeding depression via

purging or fixation of recessive deleterious mutations in small or structured populations (Glemin 2003, 2005). The contribution of genetic drift to the between-population difference in inbreeding depression may be small in the M. obovata populations, however, because the genetic diversity (expected heterozygosity) did not differ significantly between the populations, suggesting that the extent of genetic drift is similar in these populations. My study and previous ones of inbreeding depression for early survival in M. obovata are among the few that have examined intraspecific variation in inbreeding depression for early survival in mixed-mating tree species. Studies of Scots pine (Ka¨rkka¨inen et al. 1996) and Magnolia stellata (Hirayama et al. 2007) have examined such intraspecific variation and suggest that purging of the inbred load occurs at the embryonic stage. In Scots pine, environmental effects, rather than selection due to selfing, may have caused differential inbreeding depression among populations, since the levels of inbreeding in the studied populations were small (Fis =
0.085 to 0.019; Ka¨rkka¨inen et al. 1996). In the case of M. stellata, both selfing and genetic drift might have contributed to the purging of inbred load, since Hirayama et al. compared a small isolated population with a large population. In contrast to the results for embryonic survival and germination, selfed seeds were significantly heavier than outcrossed seeds in both populations. This ‘‘negative’’ inbreeding depression for seed mass has also been reported in M. stellata (Hirayama et al. 2005). The difference in seed mass between selfed and crossed seeds might be caused by a maternal-tree effect; if the amount of resources allocated per fruit for seed production is constant, then this constraint on resource allocation may result in a negative correlation between inbreeding depression for embryonic survival before seed development and that for seed mass. If differences in seed mass between selfed and outcrossed seeds affect the seeds’ viability and growth after germination, as suggested by Naito et al. (2005), then negative inbreeding depression for seed mass might mitigate the inbreeding depression expressed at the seedling stage in M. obovata. Inbreeding depression for late survival The allozyme analysis demonstrated that the inbreeding population (Misumai) and the outcrossing population (Aburayama) exhibited similar levels of inbreeding depression for late survival, with high d values. This component of inbreeding depression may have been underestimated, because application of the method of Ritland (1990) results in underestimation of inbreeding depression when the outcrossing rate varies among years, as was the case here. In the M. obovata population (Hitsujigaoka) with an intermediate level of inbreeding, inbreeding depression for late survival was also high, but lower than that in the two populations here (Ishida 2006); two inbreeding

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depression components, that for survival at the seedling stage (d = 0.38 ± 0.06; estimate from a nursery experiment ± se) and that from after this stage to maturity (d = 0.69; 95% confidence interval ranged from 0.54 to 0.75; note that this value was denoted as dl in Ishida 2006), gave a high dl value (dl = 0.81). This dl value may have been underestimated, since the conditions in the nursery may have resulted in lower inbreeding depression for survival at the seedling stage than is observed under field conditions (Ishida 2006). Accordingly, my study and previously published data on inbreeding depression for late survival suggest that this component of inbreeding depression is not correlated with the level of inbreeding at maturity, at least over the range of inbreeding levels that has been studied. These results contrast with the fact that inbreeding depression was negatively correlated with the level of inbreeding for early survival in the three populations. These results are the first to clarify the nature of the variation in the relationship between inbreeding depression and the level of inbreeding at different life stages in natural populations of a tree species. The contrast in results between late and early inbreeding depression suggests that inbreeding depression for early survival can be purged through inbreeding, whereas that for late survival is difficult to purge in this species. This idea is consistent with previous arguments from comparative studies based on interspecific or intraspecific variations in inbreeding depression (Husband and Schemske 1996; Byers and Waller 1999; Goodwillie and Knight 2006). The timing of inbreeding depression for survival has not been studied during the late life stages of M. obovata, but a previous microsatellite analysis of a natural population (Isagi et al. 2004) suggested that most selfed seedlings disappear before they become young trees. Because the seedlings exhibit inbreeding depression not only for survival, but also for growth (Ishida 2006), inbreeding depression for growth may cause severe inbreeding depression for survival before flowering in natural populations as a result of competition between selfed and crossed progeny or as a result of self-thinning, as has been shown in other tree species (Hardner and Potts 1997; Sorensen 1999). In the outcrossing population (Aburayama), the magnitude of inbreeding depression for late survival was similar to that during early survival. On the other hand, in the inbreeding population (Misumai), the magnitude of inbreeding depression for late survival was ca. 2.5 times that for early survival. These results are consistent with previous arguments that most self-fertilizing plants express the majority of their inbreeding depression late in their life cycles, whereas outcrossing plants express most of their inbreeding depression either early or late (Husband and Schemske 1996). Among long-lived plants, an equal or smaller magnitude of inbreeding depression for late survival than for early survival has been reported in many predominantly outcrossing coniferous species in nursery or plantation experiments

(Williams and Savolainen 1996; Sorensen 1999; Koelewijn et al. 1999). Inbreeding depression for late survival might have been underestimated in most of these experiments, however, because of the short study periods and mild nursery conditions used for the experiments. Previous studies of M. obovata (Ishida et al. 2003; Ishida 2006) suggest that a high level of cumulative inbreeding depression maintains reproductive traits that promote outcrossing, such as protogyny and mass flowering, despite the high frequency of self-fertilization in this species. In light of this hypothesis, self-fertilization of M. obovata as a result of geitonogamy can be regarded as a byproduct of outcrossing, as was suggested by Lloyd (1992). In this context, inbreeding depression for late survival appears to play a central role in the maintenance of reproductive traits that promote outcrossing in highly self-fertilizing M. obovata populations, because the magnitude of cumulative inbreeding depression can become high enough that outcrossing takes a selective advantage over selfing, even if the geitonogamy results in purging of inbred load for early survival (as shown in the Misumai population). Testing of this hypothesis will require further studies of the relationship between inbreeding depression and the level of inbreeding at both early and late stages in this species, using populations with a broad range in the level of inbreeding. Acknowledgments I thank T. Yahara and K. Kikuzawa for helpful discussions; K. Nagasaka, H. Yoshimaru, A. Kanazashi, T. Kawahara, and Z. M. Wang for their advice on allozyme and data analysis; H. Sato and A. Satake for their assistance with the field work; K. Nakamura, K. Tanaka, Y. Sakamoto, Y. Ishizuka, and Y. Shimada for their assistance with the allozyme analysis and germination experiment; and the Nature Center of Aburayama Forest Park for permitting my field work. I also thank K. Hirayama and the two anonymous reviewers for their invaluable comments on the manuscript. This work was supported by grants from the Forestry and Forest Products Research Institute.

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