Female and Male Fitness Consequences of Clonal Growth in A

Annals of Botany 114: 1035–1041, 2014 doi:10.1093/aob/mcu176, available online at www.aob.oxfordjournals.org

Female and male ﬁtness consequences of clonal growth in a dwarf bamboo population with a high degree of clonal intermingling

Ayumi Matsuo1,2,*, Hiroshi Tomimatsu3, Jun-Ichirou Suzuki4, Tomoyuki Saitoh5, Shozo Shibata6, Akifumi Makita2 and Yoshihisa Suyama1 1Field Science Center, Graduate School of Agricultural Science, Tohoku University, Osaki, Miyagi 989-6711, Japan, 2Faculty of Bioresource Sciences, Akita Prefectural University, Akita 010-0195, Japan, 3Department of Biology, Yamagata University, Yamagata 990-8560, Japan, 4Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan, 5Tohoku Research Center, Forestryand Forest Products Research Institute, Morioka 020-0123, Japan and 6Field Science Education and Research Center, Kyoto University, Kyoto 606-8502, Japan * For correspondence. E-mail [email protected]

Received: 19 February 2014 Returned for revision: 25 April 2014 Accepted: 8 July 2014 Published electronically: 15 September 2014 Downloaded from

† Background and Aims Although many studies have reported that clonal growth interferes with sexual reproduction as a result of geitonogamous self-pollination and inbreeding depression, the mating costs of clonal growth are expected to be reduced when genets are spatially intermingled with others. This study examined how clonal growth affects both female and male reproductive success by studying a population of a mass-ﬂowering plant,

Sasa veitchii var. hirsuta, with a high degree of clonal intermingling. http://aob.oxfordjournals.org/ † Methods In a 10 × 10 m plot, genets were discriminated based on the multilocus genotypes of 11 nuclear microsatellite loci. The relationships between genet size and the components of reproductive success were then investi- gated. Male siring success and female and male selfing rates were assessed using paternity analysis. † Key Results A total of 111 genets were spatially well intermingled with others. In contrast to previous studies with species forming distinct monoclonal patches, seed production linearly increased with genet size. While male siring success was a decelerating function of genet size, selfing rates were relatively low and not related to genet size. † Conclusions The results, in conjunction with previous studies, emphasize the role of the spatial arrangement of genets on both the quantity and quality of offpsring, and suggest that an intermingled distribution of genets can reduce the mating costs of clonal growth and enhance overall fitness, particularly female fitness. by guest on May 27, 2015 Key words: Genet size, clonal growth, inbreeding depression, reproductive success, Sasa veitchii var. hirsuta, selfing, spatial genetic structure.

INTRODUCTION fruit or seed production per flower or ramet (i.e. individual shoots) decreases with increasing genet size (Handel, 1985; Although many plant species can reproduce both vegetatively Eriksson and Bremer, 1993; Wilcock and Jennings, 1999; Wolf and sexually, the fitness consequences of clonality are not fully et al., 2000; Tarasjev, 2005; Liao et al., 2009), suggesting that understood. Clonal growth increases the size of individual female fitness of genets does not increase linearly with genet genets (i.e. a genetic individual arising from seed) and thus its re- size. One explanation is that larger genets receive proportionately productive capacity by enhancing the investment of resources less compatible pollen, because individual flowers of larger genets into sexual reproduction (Klinkhamer et al., 1989; Thompson, are more often surrounded by flowers of the same genets (Reusch, 2001). On the other hand, clonal expansion may increase the 2001; Routley et al., 2004; Wang et al., 2005; Ricardo et al., 2006). chances of pollen transfer between flowers of the same genets, Although only few studies on male success are available, male and this geitonogamous self-pollination potentially incurs both siring success has been shown to correlate negatively with genet female and male mating costs (Harder and Barrett, 1995; size in a partially self-incompatible tree, Prunus ssiori, due to Eckert, 2000) through a reduction in fitness of selfed offspring pollen discounting (Mori et al., 2009). These studies, however, (i.e. inbreeding depression) and wastage of pollen that could have focused exclusively on plant species that form distinct mono- have otherwise been used for outcrossing (i.e. pollen dis- clonal patches, probably because individual genets are easily discounting). Thus, the overall fitness of genets depends on the tinguishable in the field. Empirical evidence is still lacking for balance between these positive and negative effects of genet species in which genets are spatially intermingled, even though size (Charpentier, 2002; Jacquemyn and Honnay, 2008; such clonal architecture is predicted to ameliorate the negative Vallejo-Marıń et al., 2010). effects of clonality (Handel, 1985; Charpentier, 2002). The majority of empirical studies on the fitness consequences While genet size affects not only the quantity but also the of clonality have focused on the relationship between genet size quality of seeds produced or sired, previous work has emphasized and female reproductive success. Many studies have found that the effects of clonal architecture on selfing. More specifically,

# Crown copyright 2014. 1036 Matsuo et al. — Female and male fitness consequences of clonal growth in bamboo female selfing rates correlated negatively with local genet Field survey and sampling diversity for several clonal plants in which genets are spatially In April 2007, we established a 10 × 10 m plot in the centre intermingled (Eckert, 2000; Reusch, 2001; Albert et al., 2008). of the population (Fig. 1). Although the plot was relatively This result implies that, if genets are spatially well intermingled small compared with the entire population, an earlier study with other genets, even large genets can avoid selfing that of spatial genetic structure across the population revealed mayresult in mating costs associated with inbreeding depression. that only a few (approx. 5 %) genets extended .10 m in diameter In these studies, however, female selfing rates were compared (Matsuo et al., 2010). As previously reported for S. veitchii between patches or populations with different levels of genet (Abe and Shibata, 2012), two flowering peaks were observed diversity, while individual genets were not discriminated. during the synchronous flowering event. During the first flower- The relationship between genet size and selfing rates thus ing period (mid-May to early June), almost all florets and seeds remains largely unexplored in plant species with a high were eaten by larvae of Diptera and Lepidoptera species. Thus, degree of clonal intermingling, probably because substantial we conducted our study in the second flowering period genotyping effort is necessary to estimate selfing rates at the (mid-July to mid-August), during which approx. 40 % of seeds genet level. survived predation (A. Matsuo, unpubl. res.). After the first Here, we attempted to examine how clonal growth affects both flowering, inflorescences again emerged on culm branches female and male reproductive success in a population of mass- that appeared from the nodes of the older branches. Once flower- flowering dwarf bamboo, Sasa veitchii var. hirsuta.Thespecies ing began, all flowering culms (n ¼ 2712) in the plot is a highly clonal plant that propagates vegetatively by vigorously were mapped and tagged. For DNA analysis, leaf (or leaf Downloaded from extending rhizomes. Populations of S. veitchii var. hirsuta sheath) samples were collected from almost every flowering are likely to exhibit spatial intermingling of genets because of a culm (n ¼ 2583). Leaf samples could not be collected from relatively dispersed distribution of culms on long, creeping rhi- approx. 5 % of flowering culms because these culms did zomes (mean length between culms, 35.2 cm; range, 3–129 cm; not have any leaves. In late August, we counted the number A. Matsuo, unpubl. data). While Sasa species, including S. veitchii of seeds produced by each flowering culm (total n ¼ 19595). var. hirsuta, occasionally flower sporadically, they typically flower http://aob.oxfordjournals.org/ Approx. 8 % of the seeds produced (n ¼ 1526) were randomly once synchronously over an extensive area after long pre- sampled for paternity analysis. reproductive periods (.60 years; Ueda, 1961; Janzen, 1976; Campbell, 1985).ThemonocarpicnatureofSasa species is suit- able for examining lifetime reproductive success. In this study, we first described the spatial distribution of genets in a plot estab- Microsatellite genotyping lished in the population using 11 nuclear microsatellite loci. We then employed a field survey and paternity analysis of the seeds Total DNA was extracted from about 2 mg of freeze-dried leaf produced in order to investigate how different components of re- tissue or seed embryo using a modified cetyltrimethylammonium productive success (i.e. seed production, male siring success, bromide (CTAB) protocol (Murray and Thompson, 1980). All the by guest on May 27, 2015 female and male selfing rates) are related to genet size. samples were genotyped with11 microsatellite loci, eight of which were previously developed for other Sasa species: Sasa223, Sasa718, Sasa946, Sasa500 (Kitamura et al., 2009), BWSS-4, BWSS-5, BWSS-7 and BWSS-8 (Miyazaki et al., 2009). Three MATERIALS AND METHODS additional loci had been developed for S. veitchii: Sasa_n_03, Sasa_p_214 and CS2. Further details on the microsatellite Study system loci are provided in Supplementary Data Tables S1 and S2. We used a population of Sasa veitchii (Carrie`re) Rehder var. Multiplex polymerase chain reactions (PCRs) were carried out hirsuta (Koidzumi) S. Suzuki (Poaceae, Bambusoideae) (here- using a GeneAmp PCR System 9700 (Applied Biosystems, after S. veitchii) in northern Kyoto, Japan (35811′25′′N, Foster City, CA, USA) in a total reaction volume of 2 mL, 135849′01′′E). Plants grew densely in the understorey of a sec- containing approx. 20 ng of template DNA (dried down before ondary forest stand, dominated by broad-leaved deciduous PCRs), 1 × Multiplex PCR Master Mix (Qiagen, Hilden, trees, including Castanea crenata, Clethra barbinervis and Germany) and 0.2 mM of each primer. A pigtail sequence Quercus crispula. While the flowering cycle is not known for (GTTTCTT) was attached to the 5′ end of either the forward or S. veitchii, the species flowered in several areas of northern reverse primers to avoid scoring problems due to non-templated Kyoto from 2004 to 2007 (see Abe and Shibata, 2012 for nucleotide addition by Taq DNA polymerase (Brownstein et al., details). In early spring 2007, it was possible to see in advance 1996). Fluorescent-labelled primers were mixed with non- that our population underwent synchronous flowering by the for- fluorescent primers before they were used in PCRs. The cycling mation of flower buds. Each flowering culm bears 62.5 + 4.7 conditions were initial activation at 95 8C for 15 min; followed florets (n ¼ 30; A. Matsuo, unpubl. res.), each of which consists by 38 cycles of denaturation at 94 8C for 30 s, annealing at 57 8C of a lemma, a palea, lodicules, stamens and a pistil. Sasa veitchii for 90 s and primer extension at 72 8C for 90 s; followed by a is self-compatible and wind pollinated (S. Saito and S. Shibata, final extension step at 72 8C for 10 min. The amplified PCR pro- unpubl. res.). Although florets show some spatial separation ducts were electrophoresed on an ABI PRISM 3130xl DNA between stamens and pistils (i.e. herkogamy), we were not Analyzer (Applied Biosystems) after mixing with 10 mLof aware of any floral characters that may prevent geitonogamous Hi-Di formamide and 0.08 mL of GeneScan-500 Liz Size pollination among ramets of the same genets (e.g. synchronized Standard. We analysed the resulting data with GeneMapper 4.0 dichogamy among ramets). (Applied Biosystems). Matsuo et al. — Female and male fitness consequences of clonal growth in bamboo 1037

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F IG. 1. Position of the 10 × 10 m plot within the population of Sasa veitchii in northern Kyoto, Japan. Reproductive success of the genets for which .50 % of ﬂowering culms were found on the periphery of the plot (grey area) was not analysed. See text for details. Downloaded from

Genet identification and paternity with increasing distance from pollen donors (e.g. Messeguer et al., 2001;Rong et al., 2007). In addition, we analysed the location Genets were discriminated based on multilocus genotypes of 84 seeds sired by 20 genets with single flowering culms because (MLGs) of microsatellite loci. MLGsim 2.0 (Stenberg et al., the dispersal distance could be unambiguously determined, and 2003) was used to calculate the probability (P ) that repeated http://aob.oxfordjournals.org/ sex found that the distance of pollen dispersal in our population MLGs arose through independent sexual reproduction, taking appeared to be rather limited, with a mean distance of 1.4m into account departures from Hardy–Weinberg equilibrium 24 (Supplementary Data Fig. S1). While simulations were also run using F estimates. The probabilities were ,1.54 × 10 for IS under a different proportion of candidate males sampled (0.5) to all MLGs, so that we could reasonably assume that any repeated find the confidence level, these simulations gave essentially the MLGs resulted from clonal growth. Detection of individual same interpretation of our data. We employed the relaxed confi- genets can also be affected by somatic mutations. When MLGs dence level (80 %) for paternity assignment. While the strict confi- had a slightly distinct MLG, we considered that the variation dence level (95 %) was also used, this analysis again gave the same between them was derived from somatic mutation and grouped interpretation of our data. If the estimated pollen donor had the same by guest on May 27, 2015 them into a single genet. Specifically, when MLGs differed by genotype asthe mother, we considered the seed as selfed. Seedsthat two base pairs (i.e. one microsatellite motif) in one allele at remained unassigned to a potential male were excluded from sub- one locus but shared the same alleles at all the other loci, we con- sequent analyses that required the paternity results. The overall sidered them as belonging to the same genet and having origi- rate of paternity assignment was 59 %, and was not related to the nated from a single zygote. size of genets. The paternity of each seed was inferred using CERVUS 3.0 (Marshall et al., 1998; Kalinowski et al., 2007) which uses a likelihood-based approach to assign parentage. We used all Data analysis MLGs (n ¼ 119) found in the 100 m2 plot as candidate pollen donors. While the paternity analysis was conducted based on Genet size was defined as the number of flowering culms. The MLGs, the subsequent analyses were done at the genet level by spatial extent of individual genets (maximum linear distance summing the results for slightly different MLGs assigned to between flowering culms belonging to the focal genet) was posi- the identical genets. CERVUS assigns the paternity to a particu- tively correlated with the number of flowering culms (r ¼ 0.60, lar male if the likelihood ratio (LOD score) is large relative t ¼ 12.82, P , 0.001). Although the majority of genets probably to those of alternative males, and uses simulations to evaluate extended over spatial scales of only a few metres (Matsuo et al., the significance of LOD scores at either 95 % (strict) or 80 % 2010), our plot covered a part of the entire population and the (relaxed) confidence. In the simulations, the parameters were limited sampling may have affected the results. To reduce this used as follows: 100 000 offspring, 170 candidate male parents, bias, genets were excluded from subsequent analyses if .50 % 0.7 as the proportion of candidate males sampled, 1.0 as the propor- of the flowering culms were found on the periphery of the plot tion of loci typed, 0.02 asthe genotyping error rate (estimated on the (grey area in Fig. 1). basis of repeat genotyping of approx. 10 % of samples) and 0 as Female and male reproductive success was evaluated as seed the rate of inbreeding (based on the parental FIS value given by production (the number of seeds produced) and siring success GenAlex 6.5; Peakall and Smouse, 2006, 2012). Note that the (the number of seeds sired) of each genet, respectively. The product of the number of candidate male parents and the proportion results of the paternity assignment were used to calculate male of candidate male parents sampled is equal to the number of siring success, which includes both self-siring and outcross sampled male parents. As described above, we assumed that we siring. Since only approx. 8 % of the seeds were analysed in had sampled 70 % of the candidate males because the gene flow the paternity analysis, male siring success is a relative term. rate via pollen in wind-pollinating graminoids steeply decreases We analysed the relationships between genet size and female 1038 Matsuo et al. — Female and male fitness consequences of clonal growth in bamboo and male reproductive success using the model y ¼ axb. After the (Fig. 3). We did not find any genets excluding others from occu- datawere log transformed (or log + 1 transformed, if necessary), pying part of the plot. simple linear regressions were used to estimate the coefficients, a and b. If the regression coefficient (b) deviates from unity, the components of reproductive success are either decelerating Genet size and reproductive success (b , 1) or accelerating (b . 1) functions of genet size. We Seed production was positively related to genet size (t ¼ tested the significance of deviation of b from 1 with a t-test: 15.72, P , 0.001; Fig. 4A), and this relationship did not ts ¼ (b – 1)/sb, where sb is the standard error of the regression co- appear to be either decelerating or accelerating (b ¼ 0.992 + efficient (Sokal and Rohlf, 1995). 0.076; deviation of b from 1: t ¼ 2 0.11, P ¼ 0.22). If seed pro- The quality of offspring was evaluated as female and male duction is a decelerating function of genet size, mean seed pro- selfing rates. The female selfing rate was calculated by dividing duction per flowering culm is expected to be negatively related the number of selfed seeds by the total number of seeds analysed to genet size. However, no such relationship was found in the paternity analysis, and the male selfing rate was calculated between genet size and mean seed production per flowering by dividing the number of self-sired seeds by the total number of culm (t ¼ 21.35, P ¼ 0.18). In contrast, the relationship seeds analysed in the paternity analysis. In these analyses, we between genet size and male siring success was positive but a de- onlyincludedgenets in which the paternityof ≥8 seeds produced celerating function (b ¼ 0.374 + 0.060, t ¼ 6.21, P , 0.001; by the focal genets was assigned and ≥8 seeds sired by the focal deviation of b from 1: t ¼ 210.4, P , 0.001, Fig. 4B). As genets were detected. Using different numbers of seeds (i.e. four expected, mean siring success per flowering culm was negatively Downloaded from and six) as a cut-off value did not qualitatively affect the results. related to genet size (y ¼ 2.114 2 0.016x, t ¼ 22.25, P , Simple linear regressions were used to examine the relationships 0.05). In the analysis of selfing rates, we analysed 18 genets between genet size and selfing rates. in which the paternity of ≥8 seeds produced by those genets was assigned and ≥8 seeds sired by those genets were detected. While the female selfing rate ranged widely from 5.9to60.5%, RESULTS with a mean of 31.7 %, there was no significant relationship http://aob.oxfordjournals.org/ between genet size and female selfing rate (t ¼ 21.22, P ¼ Genet size distribution and spatial genetic structure 0.24; Fig. 4C). Male selfing rate also ranged widely from 7.7to Of 119 MLGs detected, eight had an MLG that was not very 60.0 %, with a mean of 38.7 %, and, again, there was no signifi- distinct, resulting in a total of 111 genets identified among cant relationship between genet size and male selfing rate 2583 flowering culms. Among the 111 genets, 42 genets (t ¼ 20.27, P ¼ 0.79; Fig. 4D). (38 %) were excluded from analysis because .50 % of flowering culms were found on the periphery of the plot. As a result, 69 DISCUSSION

genets were analysed in the subsequent analyses. Genet size by guest on May 27, 2015 ranged widely, from one to 191, with a mean of 29.8, and Genet size distribution and spatial genetic structure showed a leptokurtic distribution with a long right tail (median, 6.0; Fig. 2). While the distribution of genets was As commonly observed in clonal plant species (reviewed in often localized, visual inspection of the distribution map sug- Vallejo-Marı´n et al., 2010), the distribution of genet size in our gests that genets were well intermingled with each other Sasa veitchii population was leptokurtic and skewed towards lower size classes (Fig. 2). While a small number of large genets dominated the 100 m2 plot, many small genets appeared + = 69 genets to have co-occurred spatially within the matrix of larger genets 50 = 18 genets (Fig. 3). This intermingled distribution of genets may be explained in part by the leptomorph rhizome system (sensu 40 McClure, 1966)ofS. veitchii, which is characterized by long and creeping rhizomes. Culms are formed from some of the lateral buds on rhizomes, leading to a relatively dispersed distri- 30 bution of culms, compared with some other bamboo species with a patchymorph rhizome system that yields closely aggregated

Frequency culms (Makita, 1998).InS. veitchii, the dispersed distribution 20 of culms that resulted from the leptomorph rhizomes was likely to allow different genets to intermingle spatially without strongly excluding one another. The differences in such clonal 10 architecture have generally been considered to influence the extent to which genets are spatially intermingled (McLellan et al., 1997; Charpentier, 2002). 0 0 50 100 150 200 Number of flowering culms Genet size and reproductive success F IG. 2. Frequency distribution of the numberof flowering culms (i.e. genet size) among the 69 genets analysed in this study. The grey bars indicate the frequency Our study revealed that seed production linearly increased distribution of the 18 genets for which female and male selfing rates were exam- with genet size at least in the range examined here (Fig. 4A). ined. See text for details. However, most previous studies have suggested that female Matsuo et al. — Female and male fitness consequences of clonal growth in bamboo 1039

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F IG. 3. Spatial distributions of six selected genets are shown to demonstrate typical genet distributions. The sizes of the six genets (i.e. number of flowering culms) were (A) 191, (B) 88, (C) 33, (D) 20, (E) 7 and (F) 3. Black circles show the positions of flowering culms belonging to each genet, whereas the grey dots show the positions of all the other flowering culms found in the plot. reproductive success does not linearly increase with genet size size because a linear relationship between genet size and seed because female fitness per ramet is reduced (Handel, 1985; production was observed. Eriksson and Bremer, 1993; Wilcock and Jennings, 1999; In contrast, male siring success was a decelerating function of Wolf et al., 2000; Tarasjev, 2005; Liao et al., 2009). Three genet size (Fig. 4B). Although our study did not quantify the life- by guest on May 27, 2015 classes of mechanisms have been described to explain why time fitness of offspring, this result is consistent with the argument female success at the ramet level often decreases in larger that the shape of fitness gain curves usually differs between female genets. First, the number of pollen grains received per flower and male function; the male gain curve is more rapidly saturating may be lower in larger genets, because pollinators tend to visit than the female gain curve (Klinkhamer and de Jong, 2002; a smaller proportion of flowers in patches with larger floral dis- Dorken and van Drunen, 2010). As plants produce much more plays (Ohashi and Yahara, 1998; Goulson, 2000; Karron and pollen than the number of ovules, increased pollen production in Mitchell 2012). Secondly, resource competition among ramets large genets may have strengthened local mate competition with may be stronger in larger genets, because a greater number of sib pollen that reached the same stigma. Given that the opportunity ramets of the same genets may become more closely aggregated for outcrossing was not limited due to the spatial intermingling of in larger genets (Liao et al., 2009). Finally, female reproductive genets, another possible explanation is that larger genets allocate success of larger genets may be more limited by the availability proportionately fewer resources to male function, so that pollen of compatible pollen, because individual flowers of larger genets production, and thus male siring success per flowering culm, are more often surrounded by flowers of the same genets (e.g. decreases with increasing genet size. While size-dependent sex al- Wilcock and Jennings, 1999; Eckert, 2000; Barrett, 2002). location is widely observed among flowering plants (Klinkhamer Even in self-compatible species, early-acting inbreeding depres- and de Jong, 1997), the relationships between genet size and the sion can result in the abortion of selfed offspring during embryo patterns of sex allocation may depend on the spatial extent to development (Husband and Schemske, 1996) and reduce seed which ramets of the same genotypes share common resource production (Liao et al., 2009). All these explanations, how- pools. In a related species S. palmata, Saitoh et al. (2006) used ever, are more applicable to species with a clumped architecture stable isotope analyses to show that resources can readily be trans- that have been examined in previous studies. Since the first pos- located between connected ramets approx. 1 m away, although it is sibility is not applicable for S. veitchii because pollen is wind dis- not known whether translocation occurs over longer distances. persed, a plausible explanation for the linear increase in female Furthermore, male success of larger genets may have been more success with increasing genet size in our study is that the inter- underestimated than that of smaller genets in our study. While mingled distribution of genets substantially alleviated the latter many small genets were restricted to small areas in our plot, two fitness costs of clonal growth described above. Early- large genets tended to expand over the entire plot (Fig. 3). It may acting inbreeding depression may have reduced seed production thus be likely that larger genets sired proportionately more seeds to some extent, but its effect was not likely to depend on genet produced outside the plot. Even though we genotyped as many 1040 Matsuo et al. — Female and male fitness consequences of clonal growth in bamboo

as 4109 leaf or seed samplesto infer the paternity, more genotyping A 2000 effort might be necessary to obtain comprehensive estimates of P < 0·001 siring success. Whiletheseexplanations above are not mutuallyex- clusive, our experimental design was restricted to assessing the degree to which they contributed to the observed relationship between genet size and male siring success. 1000 Genet size and selfing rates Seed production Both female and male selfing rates showed no significant relationship to genet size (Fig. 4C, D). While the male selfing rate has 0 never been examined in the context of clonal growth, this result is in line with several previous studies showing negative associa- tions between local genet diversityand female selfing rate among B neighbouring patches (Eckert, 2000; Reush, 2001; Albert et al., P < 0·001 60 2008), because plenty of compatible pollen was available even in large genets due to high local genet diversity. In addition, our average estimate of the female selfing rate (31.7 %) was much Downloaded from 40 lower than that reported for another dwarf bamboo S. cernua (85.2 %), populations of which consisted of only several genets that exhibited clumped distributions (Kitamura and Kawarahara, 20 2011). The comparison of the results between these congeners sup- Male siring success ports the diminished effect of local genet diversity and thus of

clonal architecture on selfing rate. In S. veitchii, self-fertilization http://aob.oxfordjournals.org/ 0 incurs significant mating costs because of strong inbreeding depression. We estimated the magnitude of inbreeding depression 100 using Ritland’s (1990) two-generation equilibrium estimator and C found the value of inbreeding depression to be 0.85, suggesting 80 that selfed offspring will mostly be eliminated before maturity. P = 0·24 Taken together, our results, in conjunction with previous literature, emphasize the effect of the spatial arrangement of genets 60 on both the quantity and quality of offspring. Considering that individual genets of S. veitchii potentially grow over large areas for by guest on May 27, 2015 40 many decades before reproduction, it is possible that the leptomorph rhizome system has evolved to avoid the mating cost of clonal growth and enhance overall fitness, particularly female Female selfing rate (%) selfing rate Female 20 fitness. To corroborate further the effect of clonal architecture on the functional relationships between genet size and reproductive 0 success, studies that directly compare species with contrasting 100 clonal architectures or manipulate the spatial distributions of D genets will be needed.

80 P = 0·79 SUPPLEMENTARY DATA

60 Supplementary data are available online at www.aob.oxford journals.org and consist of the following. Table S1: details of the additional microsatellite locithathave previouslybeen devel- 40 oped for Sasa veitchii var. hirsuta. Table S2: characteristics of the 11 microsatellite loci. Fig. S1: pollen dispersal distance for Male selfing rate (%) Male selfing rate 20 genets with only single ﬂowering culms.

0 ACKNOWLEDGEMENTS 050100 150 200 Genet size We are grateful to Yuhei Abe for providing flowering informa- tion; Seiko Saito and Chikako Sumiyoshi for assistance in the F IG. 4. Relationships between genet size and components of reproductive field; Aya Nishiwaki and Yuji Isagi for helpful discussions; success: (A) seed production (i.e. number of seeds produced by each genet); and Mizuki Inoue for comments on the manuscript. This work (B) male siring success (i.e. number of seeds sired by each genet); (C) female selfing rate; and (D) male selfing rate. A power function was fitted to male was funded by the Japan Society for the Promotion of Science siring success because the linear regression coefficient when both axes were log- (JSPS) KAKENHI (20380092) to A. Makita and a Grant-in- transformed was significantly different from unity. Aid for JSPS Fellows to A. Matsuo. Matsuo et al. — Female and male fitness consequences of clonal growth in bamboo 1041

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