Forest Ecology and Management 256 (2008) 375–383

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Forest Ecology and Management

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Density-dependent selfing and its effects on seed performance in a tropical canopy tree species, Shorea acuminata (Dipterocarpaceae)

Yoko Naito a,1, Mamoru Kanzaki a, Hiroyoshi Iwata b, Kyoko Obayashi c, Soon Leong Lee d, Norwati Muhammad d, Toshinori Okuda e,2, Yoshihiko Tsumura f,* a Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake, Sakyo, Kyoto 606-8502, Japan b National Agricultural Research Center, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-8666, Japan c Graduate School of Agricultural Science, Tohoku University, Naruko-onsen, Osaki, Miyagi 989-6711, Japan d Forest Research Institute Malaysia, Kepong, Kuala Lumpur 52109, Malaysia e National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-0053, Japan f Forestry and Forest Products Research Institute, 1 Matsunosato, Tsukuba, Ibaraki 305-8687, Japan

ARTICLE INFO ABSTRACT

Article history: In the conservation and management practices of natural forests, sound reproduction and regeneration Received 14 December 2007 form the basis of the maintenance and viability of the tree populations. To obtain and serve biological Received in revised form 14 April 2008 information for sustainable forest management, we investigated reproductive biology and inbreeding Accepted 14 April 2008 depression in seeds of an important dipterocarp tree species, Shorea acuminata (Dipterocarpaceae), by both field and laboratory experiments. Results of parental analysis of immature and mature seeds Keywords: showed that selfing rates varied greatly, from 7.6 to 88.4% among eight mother trees, and the mean Flowering phenology overall selfing rate was 38.3%. Observed outcrossing events within a 40-ha study plot were Germination Inbreeding depression predominantly (76.5%) short-distance events with a mating distance (md) 100 m. Since the selfing Pollen flow rate sharply decreased with increase in the number of flowering conspecifics (i.e., individuals of the same Seedling establishment species) within a 100-m radius from the mother trees, the local density of flowering conspecifics appears Seed mass to be the key factor determining the outcrossing rate in S. acuminata. However, the extremely high selfing rate (88.4%) observed for one tree could not be simply explained by the low local density of flowering conspecifics. Instead, differences in its flowering phenology (its flowering peaked ca. a week earlier than most of the other examined individuals) may have severely limited its receipt of pollen from other conspecifics, and thus promoted selfing. Since there were no significant differences in the proportion of selfed progeny between immature and mature seed stages, there was no evidence of selective abortion of selfed seeds during seed development. However, the seed mass of outcrossed progeny was heavier than that of selfed progeny, and heavier seeds showed higher success rates at germination and seedling establishment. These results suggest that inbreeding depression resulted in reductions in seed mass and may reduce the fitness of selfed seeds in S. acuminata. In addition, the outcrossing rate of S. acuminata was more sensitive to low local conspecific flowering-tree densities than that of a sympatric bee-pollinated dipterocarp species with greater pollination distances. These results suggest that the management of local adult-tree densities is important for avoiding selfing and inbreeding depression in future generations, especially in a species like S. acuminata with predominantly short-distance pollination. ß 2008 Elsevier B.V. All rights reserved.

1. Introduction of many valuable timber tree species (Symington, 2004). In recent decades, many of the dipterocarp species in this region have been Lowland tropical forests in Southeast Asia are dominated by threatened by logging and other human activities that have Dipterocarpaceae (Ashton, 1982), a well-known family consisting critically reduced the numbers of their individuals and popula- tions. Accordingly, the sustainable management of remnant dipterocarp populations has become increasingly important for * Corresponding author. Tel.: +81 29 829 8261; fax: +81 29 874 3720. their sustainable use and species conservation. However, present E-mail address: [email protected] (Y. Tsumura). selective logging systems applied in Southeast Asia are not 1 Watami Food Service Company Limited, 1-1-3 Haneda, Ota, Tokyo 144-0043, necessarily sustainable, partly because tree ecology has received Japan. 2 Graduate School of Integrated Arts and Sciences, Hiroshima University, 1-7-1 limited consideration in the development of harvest–regeneration Kagamiyama, Higashi-Hiroshima 739-8521, Japan. protocols (see Sist et al., 2003). To reduce the gap between forest

0378-1127/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.foreco.2008.04.031 376 Y. Naito et al. / Forest Ecology and Management 256 (2008) 375–383 exploitation practices and conservation requirements for mixed particular, the local flowering-tree density is likely to have dipterocarp forests, more biological and ecological information extremely strong effects on the reproduction of species that about dipterocarps needs to be acquired, and incorporated in depend on weak flyers (e.g., thrips) for pollination, and moderate effective forest management policies with a sound scientific basis. effects on species pollinated by strong flyers (e.g., bees). The In the conservation and management practices of natural sensitivity or robustness of the pollination system of different forests, sound reproduction and regeneration form the basis of the species can thus be assessed by comparing the effects of local maintenance and viability of the tree populations. Previous studies flowering-tree density on their mating patterns. We chose a thrip- on mating systems and pollination ecology have shown that pollinated dipterocarp species, Shorea acuminata Dyer to make a dipterocarps are predominantly outcrossing species (Bawa, 1998; comparison with the previous results of Neobalanocarpus heimii Tsumura et al., 2003) pollinated by insects (Appanah, 1979; (King) Ashton (Naito et al., 2005), a sympatric bee-pollinated Appanah and Chan, 1981; Bawa, 1998; Momose et al., 1998). For dipterocarp species in our study site to test the validity of the predominantly outcrossing species, the maintenance of high above speculation. outcrossing rates is crucial in order to avoid inbreeding depression Thus, exploring the natural variations in mating patterns, the in future generations. However, relatively low outcrossing rates causes of such variations, and the impact of inbreeding depression have been generally reported in secondary forests (i.e., forests may provide important knowledge for predicting potential threats which has re-grown after timber harvest) compared to those in associated with changes in pollination environments due to primary forests (i.e., undisturbed forests) for dipterocarp species human disturbance such as selective logging. In this study, we (e.g., Lee, 2000; Murawski et al., 1994b; Obayashi et al., 2002). sought to obtain information on the reproductive biology and Furthermore, large variations in their outcrossing rates have been inbreeding depression in a natural population of S. acuminata,an observed among individual trees, even within a single population important tropical timber tree species in Southeast Asia. More (e.g., Fukue et al., 2007; Lee et al., 2000; Lee et al., 2006; Murawski specifically, we sought to quantify key reproductive parameters and Bawa, 1994; Murawski et al., 1994a,b; Nagamitsu et al., 2001; (i.e., flowering phenology, selfing/outcrossing rates and mating Naito et al., 2005; Obayashi et al., 2002). These findings suggest patterns) and variations of the parameters in a S. acuminata that not only ecological differences at the forest-stand level, but population in a primary forest. We then addressed the following also the fine-scale heterogeneity in pollination environments, questions. (1) How does spatial and temporal isolation of flowering heavily affect outcrossing rates of dipterocarp individuals. affect selfing rates of individual trees? (2) Are there any differences Several ecological variables have been proposed as factors that in the relationships between selfing rates and the local flowering- may affect mating patterns in tree species, e.g., spatial isolation tree density between sympatric dipterocarp species with different (Fuchs et al., 2003; Oddou-Muratorio et al., 2006), flowering pollination systems? (3) Does inbreeding affect the survival of pre- phenology (Oddou-Muratorio et al., 2006), plant density (Fran- mature seed and seed mass as a result of selective abortion or ceschinelli and Bawa, 2000) and pollinator activity (Hirao et al., inbreeding depression? (5) If so, is the seed mass correlated with 2006). Among these factors, both temporal isolation (i.e., the performance of the progeny at germination and seedling differences in flowering phenology) and spatial isolation of establishment? Finally, we discuss the implications of the results flowering (low tree density) have been proposed as causes of of the study for forest management and conservation. the observed high selfing rates of dipterocarp individuals (Fukue et al., 2007; Obayashi et al., 2002). In fact, spatial isolation (low 2. Materials and methods conspecific tree densities) has increased selfing rates in some dipterocarp species (Fukue et al., 2007; Naito et al., 2005), and 2.1. Study species flowering schedules have become important sources of temporal isolation between pollen sources and recipients in a tree species S. acuminata is a common canopy tree species that is widely (Oddou-Muratorio et al., 2006). However, to our knowledge, no distributed in mixed dipterocarp forests of Malaya, Sumatra, and previous studies have examined both effects of temporal and Lingga (Ashton, 1982). This species reproduces supra-annually in spatial isolation of flowering on the mating patterns of dipterocarp synchrony with general flowerings usually at several-year intervals. species. The shrimp-pink flowers of about 1-cm diameter are hermaphro- The impact of inbreeding depression associated with selfing is ditic, and arranged in dense, semi-pendent and paniculate also a matter of concern for predicting trajectories of future inflorescences (Appanah, 1979; Chan and Appanah, 1980). The generations. In a highly outcrossed population, lethal or deleter- flowers are thrip-pollinated (Appanah, 1979; Appanah and Chan, ious recessive alleles can be maintained within the population 1981) and their corollas are abscised the following morning (Klekowski, 1988), leading to inbreeding depression when selfing (Appanah, 1979). The plants mature seed 3.5–4 months after does occur. Inbreeding depression has been shown to affect early anthesis. Because the fruits, which have three long and two short survival (e.g., embryo survival and seedling establishment) and wings, are usually one-seeded (Chan, 1980), one fruit can be equated fitness components of progeny (e.g., seed mass) in a wide variety of with one seed. The mass of a fully developed seed (‘‘seed mass’’ of S. outcrossing plants (Husband and Schemske, 1996) and tropical acuminata in this paper corresponds to ‘‘fruit mass without wings’’) allogamous trees (e.g., Hufford and Hamrick, 2003), including a is usually ca. 0.2–0.6 g. The mature seeds, with no dormancy, dipterocarp species (Naito et al., 2005). Therefore, if the selfing rate promptly start to extend their radicles and usually complete increases, dipterocarp populations may suffer severe seed abortion expansion of the first true leaves (i.e., seedling establishment) within and/or regeneration failure as a result of inbreeding depression. 40 days of dispersal (Y. Naito, personal observation). Inbreeding depression may pose potential threats to dipterocarps in degraded forests where reproductive trees are highly isolated 2.2. Study site and focal trees and their selfing rates are increased. The mating of animal-pollinated plants is also constrained by This study was conducted in a 40-ha plot inside a primary forest the foraging ranges and patterns of pollen vectors (Ghazoul, 2005). of the Pasoh Forest Reserve (PFR), Negeri Sembilan, Malaysia. A Accordingly, mating parameters, including the degree of selfing/ detailed description of the study plot can be found in a previous outcrossing and mating distance, are likely to differ substantially study (Naito et al., 2005). Field observation and sample collection among plant species with different types of pollinators. In of seeds were carried out from September 2001 to March 2002, Y. Naito et al. / Forest Ecology and Management 256 (2008) 375–383 377

traits of seeds may vary depending on the dispersal timing of seeds (e.g., Naito et al., 2005), we collected fallen seed samples at the following seed stages: immature seeds from five focal trees (G557, G1881, G1891, G2816 and G2824) in December 2001 and mature seeds from eight focal trees (G221, G557, G601, G1026, G1881, G1891, G2816 and G2824) in January-February 2002. For seven focal trees (G557, G601, G1026, G1881, G1891, G2816 and G2824) with a long mature seed dispersal period, we used two sets of mature seeds: early-dispersed and late-dispersed (i.e., seeds that were respectively dispersed at the beginning and end of the mature seed dispersal period) for DNA analysis and the nursery experi- ment (see below). After weighing each mature seed, we used 50– 200 of the mature seed samples from each of the seven focal trees (G557, G601, G1026, G1881, G1891, G2816 and G2824) for nursery experiment. The other seed samples were brought back to the laboratory, and were used for DNA analysis. Samples of the inner bark or leaves were also collected from all 59 mature trees in the study plot for DNA analysis to determine the genotypes of all reproductive trees and to obtain genetic diversity parameters for the adult population. However, the 59 mature trees included two dead trees, from which DNA could not be extracted. Therefore, we also used a stored specimen of one dead tree for DNA extraction, and the other dead tree was treated as missing data. Thus, the genetic data for the adult population were based on 58 mature trees in this study.

2.5. DNA extraction and genotyping

Total DNA was extracted from the samples of the mature trees Fig. 1. Spatial distribution of mature Shorea acuminata trees (>30 cm dbh) within a and the progeny of eight maternal trees, using a modified CTAB 40-ha study plot in the Pasoh Forest Reserve, Malaysia. Open and filled circles method (Tsumura et al., 1996) with extraction buffer containing represent non-flowering and flowering trees, respectively. Larger filled circles 2.5% (v/v) b-mercaptoethanol. The extracted DNA was purified represent the 11 focal trees in this study. using a FastDNA Kit (Bio 101, Inc.) or High Pure PCR Template Preparation Kit (Roche Diagnostics) if the quality was not good when a general flowering/fruiting (GF2001) occurred in the study enough for polymerase chain reaction (PCR) amplification. We site. The results of flowering census data for mature trees of S. determined the microsatellite genotypes of each sample using 12 acuminata [diameter at breast height (dbh) >30 cm] suggest that primer pairs (Shc03, Shc04, Shc07, Shc09, Ujino et al., 1998; Sle280, the flowering magnitude of the S. acuminata population was Sle384, Sle392, Sle475, Sle566, Lee et al., 2004; Slu044a, Slu057, moderate (54.5% of 55 observed mature trees flowered inside the Slu175, Lee et al., 2006). plot) in GF2001 (Naito et al., 2008). Among 30 reproductive trees, PCR amplifications were performed in 7-ml reaction volumes we selected 11 focal trees (Fig. 1) to investigate individual-based containing 3.5 ml of MultiPlex mixture (QIAGEN), approximately flowering phenology, mating pattern, and seed ecology in this 0.1–1 ng of genomic DNA, and 0.2 mM of each primer (one of each study. However, three focal trees (trees G855, G1516 and G2959) pair was fluorescently labeled) using a GeneAmp PCR System did not produce enough seeds for subsequent experiments (i.e., Model 9700 (Applied Biosystems). PCR conditions were set paternity analysis and the nursery experiment, see below). These following the QIAGEN1 Multiplex PCR Handbook (QIAGEN, three trees were used solely in the evaluation of flowering 2002) with an annealing temperature of 57 8Cor608C, as phenology. appropriate, depending on the primer pairs. Fragment analysis was carried out using an ABI PRISM 3100 Genetic Analyzer 2.3. Flowering phenology (Applied Biosystems), and the size of each fragment was determined using GeneScan Analysis software and Genotyper To quantify temporal patterns of flowering intensity in individual software ver. 3.7 (Applied Biosystems). trees, we monitored the density of fallen corollas around 11 focal trees by the following seed trap method. Just before anthesis, we set 2.6. The nursery experiment 15–20 traps (plastic baskets with a ca. 0.025 m2 circular area) per tree as described in Naito et al. (2008). We collected and counted To clarify the effect of seed mass on the performance of seeds fallen corollas in the traps almost every day during anthesis, and the (i.e., germination and seedling establishment), we conducted a mean fallen corolla density (m2 day1) was calculated for each tree. nursery experiment as follows. The mature seeds (N = 1229) were individually planted, with one seed per well, in 63-well flats filled 2.4. Sample collection of seeds and adults for DNA analysis and with 100% local river sand. The flats were placed in a nursery in the nursery experiment grounds of the Forest Research Institute Malaysia in Pasoh near the study site, and were watered to maintain a sufficient moisture Immature seeds of S. acuminata are continuously dispersed level for germination/seedling establishment. Each seed was after the end of flowering, partly due to pre-dispersal seed checked for germination (here defined as the emergence of the predation, possibly due to unfruitful pollination and/or other radicle from a seed) and seedling establishment. To eliminate the causes (Naito, 2008). Since both the genetic composition and the influence of extrinsic factors on the performance of the seeds, seeds 378 Y. Naito et al. / Forest Ecology and Management 256 (2008) 375–383 which failed to germinate or establish seedlings due to an extrinsic tree. Based on the observed outcrossing events inside the plot, we cause (e.g., attack by seed-feeding insects) were excluded from examined the frequency distribution of mating distance to clarify further analysis. the outcrossing pattern (i.e., pollen dispersal pattern) of this species. To evaluate the density-dependency of the mating pattern 2.7. Data analysis in S. acuminata, the relationship between the number of the flowering conspecifics around mother trees and their respective 2.7.1. Analysis of individual-based flowering phenology selfing rates was also examined. To evaluate the effects of both To compare the flowering phenology among trees, the mean spatial and temporal isolation of flowering on selfing rates, we fallen corolla density (m2 day1) for each tree was further conducted generalized linear mixed model analysis using the data standardized to make the sum of its mean fallen corolla density of the local flowering tree density (here, we used the number of throughout the monitoring period unity. The standardized value flowering conspecifics within 99.5-m radius from the mother for each tree was then used as an index of its relative flowering trees as the index of the local flowering tree density) and flowe- intensity (RFI) on each day within the flowering period. Relative ring phenology (i.e., FOR of each mother tree) by R 2.4.1 (The R flowering intensity within the population (RFIPOP) on given days Development Core Team: glmmML package). was expressed as the mean RFI value for all 11 focal trees. To examine the level of flowering synchrony between 2.8.2. The effect of cross type on seed mass and the effect of seed mass individual trees and the population, a flowering overlap ratio on performance of seeds

(FOR) was calculated for each focal tree using the following the To investigate the effect of cross type (selfing vs. outcrossing) on equation, the mass of mature seeds, a linear mixed model analysis was performed using the data derived from the DNA analysis of mature XD seeds. The model included cross type and dispersal period (early vs. F ¼ minðRFI ; RFI Þ; (1) OR ik POPk late dispersal) as fixed effects, and mother tree as a random effect. k¼1 The effect of seed mass on the performance of seeds was also where D is the monitoring period of flowering trees (D = 55 days), investigated by fitting a nominal logistic curve for germination and

RFIik and RFIPOPk are the relative flowering intensities for individual seedling establishment to the results obtained from the nursery i (1 i 11) and the population on the kth day, respectively. FOR experiment. Only data on germinated seeds were subject to the values (0 FOR 1) of zero and one indicate no overlap and perfect analysis of seedling establishment. All the standard statistics were overlap of flowering phenology, respectively. calculated using JMP5.0 software (SAS Institute Inc., 2002).

2.8. Genetic diversity 2.8.3. Comparison of the sensitivity of the mating pattern to local flowering-tree densities with that of another dipterocarp species Genetic diversity parameters of the adult population were To compare the sensitivity of S. acuminata’s mating pattern to estimated using genotype data of the mature trees. The observed the local conspecific flowering-tree density with the sensitivity of

(Ho) and expected (He) heterozygosity, and the paternity exclusion other sympatric dipterocarp species, data from a paternity analysis probability (PE) for each microsatellite locus was estimated using of N. heimii seeds (Naito et al., 2005) were reanalyzed in this study. CERVUS 2.0 software (Marshall, 2001). The coefficient of inbreed- Since no phenological data on the flowering of N. heimii trees was ing (FIS) in the adult population was also calculated using the obtained at the time of the cited study, the effective pollen donors equation: FIS =1 (Ho/He)(Nei, 1977). The statistical significance were considered to be the flowering trees in this analysis. of the deviation of the FIS value from Hardy–Weinberg expectation at each locus was assessed by randomized tests using FSTAT 3. Results (Goudet, 2001). 3.1. Flowering phenology 2.8.1. Paternity assignment and direct estimates of selfing rates and pollen flow There was an 11-day spread in the date of peak flowering, and

The paternity of each seed was assigned by means of simple FOR ranged from 0.543 to 0.881 (mean: 0.768) among the 11 focal exclusion, based on the multilocus genotypes of the 30 flowering trees (Fig. 2). Flowering was generally highly synchronized, except trees. If any progeny lacked any suitable pollen donor candidates, for the trees G601 and G1516, both of which showed earlier peak we assumed its pollen donor was located outside the study plot. If flowering than the other trees, with relatively low FOR values. any progeny had two or more pollen donor candidates, we inferred paternity based on maximum likelihood paternity assignment 3.2. Genetic diversity and polymorphism of microsatellite markers (Marshall et al., 1998) using the computer program CERVUS 2.0 (Marshall, 2001). The allele frequency among the mature trees was We detected 3–13 alleles per locus and 101 alleles in total at the used for this simulation, instead of the allele frequency among the 12 microsatellite loci among 58 mature trees examined (Table 1). entire sample. The simulation parameters were as follows: 10,000 The observed and expected heterozygosity for the 12 loci averaged cycles, 30 candidate parents (the number of all flowering trees in 0.674 and 0.676, respectively. A significant deviation of the FIS the study area), 100% for both the proportion of candidate parents value from Hardy–Weinberg expectation was observed at one sampled, 0% for the rate of typing error, and 80.0% for the locus, Slu175 (P < 0.05). The total paternity-exclusion probability confidence level. Using this approach for paternity assignment, (PE) over these loci was 0.9997, indicating that they can be used as proportions of self-fertilized (selfed) progeny were determined high-resolution markers for parentage analysis. Both the deficit in directly for seeds (immature seeds and mature seeds including the heterozygosity at locus Slu175 and the frequent mismatching early- and late-dispersed seeds) in each family, and the proportion observed in the homozygous genotypes of the progeny with those of selfed progeny was compared between immature and mature of their mother at two loci for particular maternal trees (G 557, seed stages using R 2.4.1 (The R Development Core Team: glmmML G1891 and G2816 at Slu175 and G557 at Sle566) suggest the and MASS packages). The proportion of selfed progeny to the total existence of null alleles at these loci. Therefore, we assigned pollen seeds in each family was regarded as a selfing rate for each mother parents assuming null alleles when the genotypes of the progeny Y. Naito et al. / Forest Ecology and Management 256 (2008) 375–383 379

(P > 0.05), we pooled the data of two mature seed stages, and the proportion of the selfed progeny was compared between immature and mature seed stages for five trees (G557, G1881, G1891, G2816 and G2824). The proportion of the selfed progeny was higher in mature seed stage than that in immature seed stage (P =0.016).

3.4. Pollen flow and mating pattern

We were able to assign pollen donors to 46.8% of the outcrossed progeny, and we assumed that the pollen donors of the rest of the outcrossed progeny were located outside the study plot. Most (76.5%) of the detected outcrossing events inside the plot were short-distance matings, with mating distance 100 m (Fig. 3). Furthermore, the selfing rate sharply decreased with increases in the number of flowering conspecifics within a 100-m radius of the mother trees, i.e., local density of flowering trees (Fig. 4). The result of generalized linear mixed model analysis showed that a model with only the local flowering tree density had the lowest AIC, and the local flowering tree density had the negative effect on the selfing rate (P = 0.00021).

3.5. The effects of cross type on seed mass and of seed mass on seed performance

Seed mass was affected by cross type and dispersal period, but we did not detect a significant interaction between these factors (Table 3). The rates of both successful germination and seedling establishment increased with increases in seed mass class (Fig. 5). The results of a nominal logistic fit for germination showed that the effects of both seed mass and mother tree on germination were statistically significant (Table 4). In contrast, only the effect of seed mass was significant for seedling establishment (Table 4).

3.6. Comparison of the sensitivity of the mating pattern to local flowering-tree densities with that of another dipterocarp species

The density of the effective pollen donors of N. heimii within the 40-ha study plot was 0.58 tree/ha, suggesting that the flowering density of N. heimii was even lower than that of S. acuminata in GF2001. However, despite the apparently low local flowering density in N. heimii it maintained a substantially lower selfing rate than that of S. acuminata (Fig. 6).

Table 1 Profiles of 12 microsatellite loci detected in 58 mature trees of Shorea acuminata Fig. 2. Temporal changes in the relative flowering intensity (RFI) for 11 focal trees of S. acuminata. Mean RFI values for 11 focal trees were used as an index of flowering Locus Number of alleles Size range Ho He FIS PE phenology of the whole population (‘‘Population’’). The peak flowering is Shc03a 3 123–125 0.345 0.355 0.028 0.151 highlighted with an asterisk. The date of peak flowering and the flowering Shc04a 13 79–132 0.877 0.834 0.052 0.663 overlap ratio (FOR) of each tree is also shown. For methods used to calculate RFI and Shc07a 8 154–182 0.793 0.812 0.023 0.626 FOR, see text. Shc09a 10 169–195 0.759 0.745 0.019 0.525 Sle280b 5 108–116 0.517 0.508 0.018 0.274 b were homozygous at loci Slu175 and/or Sle566. Prior to paternity Sle384 8 198–268 0.772 0.727 0.062 0.490 Sle392b 8 172–186 0.741 0.758 0.022 0.531 assignment, we excluded progeny samples whose genotypes did Sle475b 10 126–220 0.483 0.474 0.019 0.292 not match those of the assumed mothers because these progeny Sle566b 12 61–114 0.828 0.818 0.012 0.636 were considered to have dispersed from other fruiting trees. As a Slu044ac 13 152–216 0.845 0.794 0.064 0.598 c result, 7.8% of the total seeds examined (N = 688) were excluded Slu057 4 109–120 0.586 0.548 0.069 0.248 Slu175c 7 190–214 0.537 0.742 0.276* 0.511 from further analysis. Average 8.42 0.674 0.676 0.003 0.462 3.3. Variation of selfing rates between individuals and seed Total 101 0.9997 developmental stages The observed number of alleles, allele size range, observed (Ho) and expected (He)

heterozygosity, inbreeding coefficient (FIS) and paternity exclusion probability (PE) Selfing rates varied greatly, from 7.6 to 88.4%, and the mean of each locus is shown. The total PE over these loci was 0.9997. a Ujino et al. (1998). overall selfing rate was 38.3% among the eight mother trees b Lee et al. (2004). (Table 2). Since there was no statistically significant difference in the c Lee et al. (2006). proportions of selfed progeny between the two mature seed stages * Significant deviation from Hardy–Weinberg expectation (P < 0.05). 380 Y. Naito et al. / Forest Ecology and Management 256 (2008) 375–383

Table 2 Proportions of selfed progeny in immature and mature seeds of eight S. acuminata trees

Mother tree Seed developmental stage

Immature Mature Total

Early dispersal Late dispersal Total

G221 – 44.2 (19/43) 44.2 44.2 (19/43) G557 5.0 (1/20) 20.6 (7/34) 32.3 (10/31) 26.2 21.2 (18/85) G601 – 88.6 (31/35) 88.2 (30/34) 88.4 88.4 (61/69) G1026 – 51.4 (18/35) 40.0 (14/35) 45.7 45.7 (32/70) G1881 47.1 (16/34) 41.2 (14/34) 54.5 (18/33) 47.8 47.5 (48/101) G1891 19.2 (5/26) 40.6 (13/32) 45.7 (16/35) 43.3 36.6 (34/93) G2816 20.0 (4/20) 37.9 (11/29) 28.1 (9/32) 32.8 29.6 (24/81) G2824 16.0 (4/25) 5.9 (2/34) 3.0 (1/33) 4.5 7.6 (7/92) Total 24.0 (30/125) 41.2 (96/233) 42.1 (98/233) 46.9 38.3 (243/634)

The numbers of selfed progeny (left) and those of the progeny examined (right) are shown in parentheses.

Fig. 4. Relationship between selfing rate and local density of flowering conspecifics (within a 100-m radius of the mother trees) in S. acuminata. Three focal trees (trees G221, G557 and G601) located at the margin of the study plot (i.e., less than 100 m far from the edge of the study plot) were excluded from this analysis.

Fig. 3. Frequency distributions of (a) mating distance based on the observed outcrossing events inside the plot and (b) distance from the mother trees to the potential mates inside the plot for eight trees of S. acuminata.

4. Discussion

4.1. Population outcrossing rate

The estimated outcrossing rate for the S. acuminata population Fig. 5. Relationships between seed mass (in five classes) and success rate of was generally lower than those observed in primary forests for germination and seedling establishment in the progeny of seven S. acuminata trees. other dipterocarp species (Fukue et al., 2007; Kenta et al., 2004; Kitamura et al., 1994; Lee, 2000; Lee et al., 2000; Lee et al., 2006; Table 4 Murawski et al., 1994b; Nagamitsu et al., 2001; Naito et al., 2005; Results of a nominal logistic fit for germination and seedling establishment testing the effects of seed mass, dispersal period and mother tree by likelihood ratio test

Table 3 Life stage Source of variation d.f. x2 P Results of generalized linear mixed model analysis testing the fixed effects of cross type, dispersal period and their interaction on mass of mature seeds in S. acuminata Germination Seed mass 1 174.084 0.0000 Dispersal period 1 2.566 0.1092 Factor d.f. SS FP Mother tree 6 80.769 0.0000

Cross type 1 0.0584 16.09 0.0070 Seedling establishment Seed mass 1 16.113 0.0001 Dispersal period 1 0.0353 9.73 0.0206 Dispersal period 1 0.014 0.9068 Cross type dispersal period 1 0.0013 0.36 0.5705 Mother tree 6 7.357 0.2891 Y. Naito et al. / Forest Ecology and Management 256 (2008) 375–383 381

Obayashi et al., 2002) and other tropical tree species (Ward et al., pollinators of S. acuminata in the same forest in previous studies 2005). Previous studies have shown that the outcrossing rates of were short-distance-flyer, thrips (Appanah, 1979; Appanah and dipterocarps in primary forests usually exceed 80%, but the Chan, 1981), also supports this hypothesis. outcrossing rate we obtained for S. acuminata was only 61.7%. Since However, the extremely high selfing rate of the tree G601 previous studies suggest that densities of flowering conspecifics (88.4%) could not be simply explained by a low local density of affect outcrossing rates in some dipterocarp species (e.g., Lee, flowering conspecifics. In fact, the local density around G601 was 2000; Murawski et al., 1994b; Obayashi et al., 2002), the low relatively high (there were at least four flowering conspecifics outcrossing rate of S. acuminata is likely to be due to the very low within a 100-m radius of the tree) although it was located at the density of flowering conspecifics (0.75 tree/ha) in the population margin of the plot and was thus excluded from Fig. 4 because of a we examined. lack of data on the exact local flowering-tree density around it. In addition to the density of flowering conspecifics, the flowering However, as shown in Fig. 2, the flowering peak of G601 occurred magnitude of the whole plant community (i.e., the magnitude of ca. a week earlier than in most of the other trees, and it had the general flowering (GF)) can also affect the mating pattern of ‘‘GF lowest FOR value among the eight trees examined to analyze the type’’ plants by altering the composition of the pollinator commu- mating pattern in this study. Even though the result of generalized nity. Sakai et al. (1999) and Sakai (2002) hypothesized that a GF, linear mixed model analysis showed that the effect of flowering community-level flowering event over a wide range of plant taxa phenology on selfing rate was not significant, this lack of including dipterocarps, is an adaptive strategy to attract more significance rather seems to be due to the very small variations effective and larger numbers of pollinators, thereby promoting in FOR values among the six trees used for that analysis. Therefore, cross-pollination (i.e., ‘‘promotion-of-pollination hypothesis’’). And the difference between its flowering phenology and that of other in fact, changes in the composition and abundance of pollinators of flowering conspecifics may have severely limited its receipt of dipterocarps have been observed between two GF events with outcross pollen, and thus increased its rate of seed production different flowering magnitudes in a forest in Sarawak (intensive GF through selfing (i.e., self-pollination and geitonogamy). Although in 1996 vs. less intensive GF in 1998; Kenta et al., 2004). Notably, other factors have also been reported to influence outcrossing rates Dipterocarpus tempehes was mainly pollinated by Apis dorsata in (e.g., the degree of self-incompatibility and canopy position: 1996 but by moths in 1998 (Kenta et al., 2004) and the abundance of Patterson et al., 2004), the degree of spatiotemporal isolation from A. dorsata was higher in 1996 than in 1998 (Itioka et al., 2001). other flowering conspecifics may be the main determinant of the Therefore, the lower outcrossing rate of S. acuminata observed in this mating pattern and selfing rate of this species. study than reported rates for other dipterocarps may be at least partly due to the small magnitude of the sporadic GF at the time of 4.2.1. Inbreeding effect on seed our study compared to the other GF events in which the previous There was no evidence of selective abortion of selfed seeds studies were undertaken, e.g. the mass GF at the time of the study by during seed development because the proportion of selfed progeny Nagamitsu et al. (2001) in 1996 at the same study site (Numata et al., in mature seed stage was higher than that in immature seed stage 2003). Further comparative studies under different pollination (Table 2). Intense selection on selfed embryo might occur at mass conditions (e.g., with differences in the magnitude of GF and density abortion of tiny seeds immediately after anthesis though these of flowering conspecifics) are needed to clarify more thoroughly the seeds were barren and could not be subjected to DNA analysis (Y. effects of pollination conditions at the community and/or popula- Naito, personal observation). However, the significant effect of tion levels on outcrossing rates of these GF type tree species. cross type on seed mass (Table 3) indicates that there was a significant difference in the mature seed mass between selfed and 4.2. Mating pattern, individual selfing rates and causes of their outcrossed progeny. Moreover, heavier seeds showed better variation performance than lighter seeds at germination and seedling establishment (Fig. 5 and Table 4) and, in particular, only seed mass Outcrossing events in S. acuminata predominantly consisted of had a significant effect on seedling establishment (Table 4). Since short-distance mating events, with mating distances (md) 100 m the seed mass of selfed progeny was lighter overall than that of the (Fig. 3). Together with the finding that nearly 40% of the total seeds outcrossed progeny, failure to germinate or establish may be more were produced through selfing (md = 0 m), this indicates that long- likely among lighter, selfed seeds than among heavier, outcrossed distance mating (md > 100 m) was far less frequent than short- seeds. These results suggest that inbreeding depression occurs in distance mating (which accounted for at least 60.4% of the total the early life stages of S. acuminata. Inbreeding depression has also mating events examined) in this species. Its mating pattern was been shown to reduce the seed mass and the proportion of clearly different from patterns observed by Fukue et al. (2007) for S. surviving selfed progeny up to seedling stage in two other tropical leprosula and Kenta et al. (2004) for Dipterocarpus tempehes, both of tree species, including another dipterocarp (Hufford and Hamrick, which showed much higher outcrossing rates with much higher 2003; Naito et al., 2005). These findings accord well with the frequencies of long-distance mating (ca. 70% and 60% of out- previous finding that substantial inbreeding depression occurs in crossing events, respectively) than S. acuminata. However, in the the early life stages of outcrossing plants (Husband and Schemske, cited studies the mating parameters of S. leprosula and D. tempehes 1996), and imply that allogamous tropical tree species may suffer were inferred from data acquired from seedling populations, and substantial inbreeding depression early in their life cycles. thus the outcrossing rates and/or pollen dispersal patterns may have been biased by the influence of inbreeding depression at the 4.3. Comparison of the sensitivity of the mating pattern to local stage of seedling establishment (Kenta et al., 2004). flowering-tree densities with that of another dipterocarp species Since the selfing rate was strongly affected by the local flowering-tree density within a 100-m radius of the mother tree In contrast to S. acuminata, relatively low selfing rates were (Fig. 4), high opportunities for short-distance outcrossing appear to maintained, despite the low local density of flowering conspecifics, be essential for maintaining high outcrossing rates in this species. in the sympatric bee-pollinated dipterocarp species, N. heimii Furthermore, the observed mating pattern (Figs. 3 and 4) suggests (Fig. 6; cf. Fig. 4). This result suggests that N. heimii has wider that short-distance flyers may have been the main pollinators of S. ranges of pollen exchange than S. acuminata, mediated by stronger acuminata in this reproductive event. The finding that the main flyers (bees rather than thrips), and thus its outcrossing rate is less 382 Y. Naito et al. / Forest Ecology and Management 256 (2008) 375–383

our nursery experiment; Drs. Y. Suyama, S. Ueno, M. Ohtani, Y. Tsuda, Y. Takeuchi and Ms. Y. Taguchi for their helpful advice on DNA and statistical analysis; Mr. E.S. Quah, Dr. K.K.S. Ng, Mr. A. Hussein, Mr. A. Nyak and all the local staff involved in this project for their assistance during sample collection and/or the nursery experiment; Ms. D. Mariam, Ms. Nor Salwah A.W., Ms. Nurl Hudaini Mamat and members of the Genetic Lab at FRIM for their assistance in the laboratory; an anonymous reviewer who gave us invaluable comments on statistical analyses and greatly helped improve this manuscript. This study was a part of a joint research project of FRIM, Universiti Putera Malaysia, and National Institute for Environmental Studies of Japan (Global Environment Research Program supported by the Ministry of Environment in Japan, Grant Fig. 6. Density-dependency of the selfing rate of a sympatric, bee-pollinated no. 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