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 reproduc- tion 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 micro- satellite 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 dis- counting). 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-Marı´n 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