Generalist Pollinators in the Dioecious Shrub Rhus Trichocarpa Miq

Plant Species Biology (2009) 24, 215–224 doi: 10.1111/j.1442-1984.2009.00258.x

NOTES AND COMMENTS Generalist pollinators in the dioecious shrub Rhus trichocarpa Miq. (Anacardiaceae) and their role in reproductive successpsbi_258 215..224

SHUHEI MATSUYAMA,* NAOYA OSAWA† and MICHINORI SAKIMOTO‡ *Laboratory of Silviculture, Division of Forest and Biomaterials Science, †Laboratory of Forest Ecology, Graduate School of Agriculture and ‡Laboratory of Forest Species and Ecosystem Conservation, Field Science Education and Research Center, Kyoto University, Kyoto 606-8503, Japan

Abstract

We examined the floral display size and potential pollinators of female and male Rhus trichocarpa in northern Kyoto, Japan, in June 2005. The entomophilous pollination system comprised 431 insects and one spider belonging to 124 species. Most pollinators were non-social bees and occasional Diptera and Coleoptera, whereas eusocial bees were not dominant in the pollination system. Male-biased frequencies were observed in the eusocial bees and in some of the non-social bees, probably because they are sensitive to the larger male floral rewards. A pollinator introduction experiment confirmed that male- biased, unbiased and infrequent non-social bees can pollinate R. trichocarpa, indicating that non-social bees can contribute to fruit set. The results suggest that there are likely to be two different functional groups of generalist pollinators: (i) the majority of the pollinators may contribute to reproductive success through unbiased and occasional visits; and (ii) a minority group of eusocial bees may contribute to reproductive success through male-biased visits. Keywords: dioecy, floral display size, generalist pollinator, Rhus trichocarpa, visitation frequency. Received 1 May 2009; accepted 13 July 2009

Introduction Pollinators, including generalists, usually use floral rewards from plants. Several studies have shown that pol- Interaction with pollinators is a determining factor in linators will visit plants with larger floral rewards more entomophilous plant reproductive success; reproductive frequently than those with smaller rewards (e.g. Huang success depends on the availability of resources for et al. 2006). However, the frequency of visits to focal allocation into plant reproductive organs and on the effec- plants differs among pollinator species (Ågren et al. 1986; tiveness of pollen transfer by animal vectors. Most Farwig et al. 2004; Voigt et al. 2005). Some studies indicate entomophilous interaction studies have focused on the that individual plant species enhance their reproductive relationships between plants and specialist pollinators success through interactions with many species of (e.g. Kevan & Baker 1983) or frequent generalist pollina- frequent and infrequent generalist pollinators (e.g. tors, such as eusocial bees (e.g. Mitchell et al. 2004; Hira- Schemske & Horvitz 1984; Dukas 1987). bayashi et al. 2006). Recent theoretical studies suggest that Many dioecious plant species are believed to be polli- plants may favor generalist as well as specialist pollinators nated mainly by generalist pollinators, such as eusocial (Waser et al. 1996; Aigner 2001; Sargent & Otto 2006). and solitary bees, flies, other Diptera and Coleoptera (e.g. However, generalist pollinator interactions with plants Opler et al. 1980; Muenchow 1987). In addition, dioecious are less well documented. plant pollination is believed to be more efficient than that in hermaphrodite species (Sutherland & Delph 1984). Correspondence: Shuhei Matsuyama Thus, dioecy and/or the generalist-dominant pollinator Email: [email protected] syndrome contribute to highly efficient pollination

© 2009 The Authors Journal compilation © 2009 The Society for the Study of Species Biology 216 S. MATSUYAMA ET AL. success (i.e. reproductive success). However, the precise with numerous small flowers (Ohwi & Kitagawa 1992). mechanisms elevating reproductive success in these cases Most flower corollas are <10 mm wide and <10 mm tall (S. are unclear because solitary bees, flies, other Diptera and Matsuyama, 2005). In the study area flowering occurs in Coleoptera are believed not to transfer pollen as effec- early June and fruits mature in October (Matsuyama & tively as eusocial bees (e.g. bumble bees), which some Sakimoto 2008). The sexes of R. trichocarpa flowers can dioecious plants use as their preferred pollinators in be distinguished by color; male flowers are yellowish nature (Thomson et al. 1982; Dukas 1987; Herrera 1989). and female flowers are greenish (S. Matsuyama, unpubl. Several empirical studies have demonstrated that the data, 2001). Male flowers produce both pollen and pollination systems of dioecious species are characterized nectar, whereas female flowers produce only nectar in by biased pollinator visits to male plants because of R. trichocarpa (S. Matsuyama, unpubl. data, 2005). elevated rewards in their flowers (e.g. Ågren et al. 1986; Bierzychudek 1987; Elmqvist et al. 1988). However, Visitor fauna and visitation frequency extremely male-biased visits in dioecious plants may lead to pollen loss and to a reduction in reproductive success In 2005, we randomly selected 20 flowering plants (9 (Charlesworth 1993). Hence, a non-bias in the frequency of male, 11 female) and recorded visitation fauna, visitation visits may play a certain role in the reproductive success of frequency, floral display size and fruit set. dioecious plants in addition to the frequency of visits. To examine visitor fauna and visitation frequency in R. Rhus trichocarpa (Anacardiaceae) is a common ento- trichocarpa, flower visitors to female (n = 11) and male mophilous dioecious shrub that favors disturbed sites in plants (n = 9) were captured on fine weather days in the temperate zone of Japan (Kamitani et al. 1998; Osada June 2005 (from nine plants on 9 June, six plants on 10 2005). Flowers of Rhus spp. are visited by several species June and five plants on 14 June). Fine weather days were of bees, flies and other insects (Kato et al. 1990), suggest- chosen because there is a negative effect of rain on the ing that many generalist pollinators influence the repro- number of visitors. For each plant, one reproductive ductive success of the plants. current-year shoot with open flowers was selected and The objectives of the present study were to determine all arthropods on inflorescences of the shoot were caught the effects of visits by many generalist pollinators on the over 15 min. For the first 12 min all of the visitors were reproductive success of R. trichocarpa in a primary beech caught with a net, after that an aspirator was used for the forest in Japan by: (i) identifying all potential pollinators to last 3 min. This sampling was repeated five times per the species level; (ii) evaluating floral display size and fruit day for the same shoot on each plant, from approxi- set; and (iii) evaluating the pollination efficiency of selected mately 08.00–13.00 hours at 45-min intervals. potential pollinators using an introduction experiment. All visitor samples were pinned and labeled with com- plete census data (date, locality and flower type). Visitors were classified to the species level where possible. Three Materials and methods hundred and thirty-eight individuals, 78.2% of the total, were identified to species. The remaining individuals (95) Study site and target species were assigned to higher taxa (generally family). All speci- The study was conducted in 2005 at the Asiu Forest mens were deposited in the Laboratory of Silviculture, Research Station of Kyoto University in the northern part Graduate School of Agriculture, Kyoto University. of Kyoto Prefecture, western Japan (35°18′N, 135°43′E; One dataset of visits per 15 min for all visitor species 750 m a.s.l.). The mean annual air temperature and annual and each plant (5 collection times ¥ 124 species ¥ 20 precipitation at a nearby weather station are 11.9°C and plants = 12 400 data points) was constructed. The average 2298.3 mm, respectively (Kyoto University Field Science visitation frequency per 15 min was calculated for each Education Research Center 2007). The study area is a species and for the total species and was compared among natural conifer/hardwood forest dominated by the ever- arthropod orders. For each order, the average visitation green conifer Cryptomeria japonica and the deciduous frequency was compared among species captured on broadleaf species Fagus crenata, Acer sieboldianum and female and male plants. The difference between plant Quercus crispula (see Hirayama & Sakimoto 2003). sexes was examined as the average visitation frequency Rhus trichocarpa is native to the mountains and foothills for the total species, and for the species captured on both of north-eastern Asia: Japan, including the southern female and male plants. Kuriles, Hokkaido, Honsyu, Shikoku, Kyusyu and Okinawa; Korea; and China (Ohwi & Kitagawa 1992). It is Floral display size a deciduous shrub approximately 4 m in height in the study area (Matsuyama & Sakimoto 2008). The inflores- To evaluate the display size of female and male flowers cence forms from axillary buds on current-year shoots, we calculated the number of flowers per plant by multi-

© 2009 The Authors Plant Species Biology 24, 215–224 Journal compilation © 2009 The Society for the Study of Species Biology POLLINATION SYSTEM OF RHUS TRICHOCARPA 217 plying the number of reproductive current-year shoots introduced onto female inflorescences enclosed in bags per plant by the number of inflorescences per shoot and and fruiting of the bagged inflorescences was monitored. by the number of flowers per inflorescence for the plants Thirty inflorescences from 15 shoots from five female sampled in 2005. The number of reproductive current- plants were selected on 25 May 2005 (before flower year shoots per plant was counted for each sampled opening) and enclosed in mesh bags (82.9 mm mesh size), plant. Three reproductive current-year shoots were har- which prevented visitors from accessing the flowers. vested from each plant except for one female, from Several species of flower visitors were captured on male which only one was harvested. As a result, we sampled a plants within 15 min on 3, 4, 9, 10, 14 and 17 June, follow- total of 58 current-year shoots (31 female and 27 male). ing flower opening. The captured bees were introduced The number of inflorescences per shoot was counted for into the mesh bags as soon as possible. One to four bees each sample shoot and we calculated the mean number were introduced into each bag to avoid fruiting failure as of inflorescences per shoot for each plant. The number of a result of too little pollen on the captured bees for polli- flowers per inflorescence was counted for all inflores- nation. Bee species were introduced into bags without cences (237 female and 246 male) on each sampled shoot, identification to species because specific identification was and we calculated the mean number of flowers per inflo- difficult in the field. rescence for each sampled plant. The numbers of flowers At the end of September, 28 bags were collected (two per female and male plant were calculated using the bags had fallen before collection). We harvested bagged following equation: number of flowers per plant = inflorescences containing fruits, unpollinated flowers, number of reproductive current-year shoots ¥ number aborted fruits and dead bees. We counted the numbers of of inflorescences per shoot ¥ number of flowers per fruits per inflorescence, aborted fruits and the number of inflorescence unpollinated flowers in each bag. The number of flowers was calculated by summing the total fruits per inflores- Fruit set cence, aborted fruits and unpollinated flowers per bag. Bagged dead bees were identified mainly by head and To estimate fruit set, three infructescences were harvested wing features. Data from bags that included single species from three current-year shoots on each fruiting female. We were collected. We analyzed the data for species that were counted the total number of fruits per infructescence and captured on both female and male plants in the survey of measured the length of the main axis of each infructes- visitation fauna. cence. To estimate the number of flowers per infructescence, the number of flowers and the length of the main Data analysis axis were obtained for each sampled female inflorescence in 2005 (n = 237). We modeled the relationship between the The differences between the sexes in the number of number of flowers per inflorescence and the length of flowers per plant were analyzed using a generalized the inflorescence main axis. The number of flowers per linear model (glm in the R package BASE) with a Poisson infructescence was calculated from the length of the main distribution and a log-linked function. axis of an infructescence based on the relationship between To test differences among orders and species in visita- the length of the main axis of an inflorescence and the tion frequencies per 15 min, we used a Kruskal–Wallis number of flowers per inflorescence (log y = 3.820 ¥ non-parametric test. Multiple comparisons were con- log x + 0.101, where x is the length of the inflorescence ducted using Kruskal–Wallis non-parametric tests with main axis and y is the number of flowers per inflorescence; Bonferroni’s method. The differences between the plant d.f. = 225, t = 181.900, P < 0.001 using a generalized linear sexes in the visitation frequencies for total and individual model with a Poisson distribution, a log-linked function species were analyzed using a Mann–Whitney U-test. In and a random effect for difference within the individual). addition, we used a Kruskal–Wallis non-parametric test In the present study, we define fruit set as the number of for preliminary analyses of the differences among sam- fruits per infructescence divided by the estimated number pling hours for visitation frequency per 15 min. of flowers per inflorescence because we could not count all All statistical analyses were carried out using R 2.4.0 (R flowers on an inflorescence without destroying the inflo- Development Core Team 2006). rescence in the field because of the numerous and small flowers densely attached to the inflorescence. Results Potential pollinator introduction Visitor fauna and visitation frequency To examine the function of visitors from the genus Lasio- A total of 432 individuals from 124 species in five orders glossum (Halictidae) and the genus Andrena (Andrenidae) were collected from male and female flowers of R. tri as pollinators, visitors captured on male plants were chocarpa (Table 1). Eighty-four and 66 species visited

Table 1 Species visiting female and male Rhus trichocarpa at the Asiu Forest Research Station, Kyoto, in June 2005

Class Order Family Species Male Female

ARACHNOIDEA Araneae Araneidae Araneidae sp. 0 1 INSECTA Hemiptera Cicadellidae Cicadellidae sp. 0 1 Psyllidae Trioza nigra Kuwayama 1 0 Psyliidae sp. 0 1 Aphididae Aphididae sp. 1 0 Miridae Gigantomiris jupiter Miyamoto et Yasunaga 1 0 Lygocoris sp. 0 1 Psallus sp. 2 0 Coleoptera Carabidae Colpodes lampros Bates 1 1 Histeridae Niponius osorioceps Lewis 0 1 Trypeticus fagi Lewis 0 1 Staphylinidae Priochirus japonicus Sharp 1 0 Elateridae Ampedus aureovestitus aurovestitus Kishii 5 0 Ampedus tenuistriatus Lewis 1 1 Ampedus vestitus vestitus Lewis 1 0 Ectinus sericeus sericeus Candeze 2 0 Melanotus correctus correctus Candeze 1 0 Erotylidae Tritoma niponensis Lewis 0 1 Cephaloidae Cephaloon pallens Motschulsky 2 2 Lagriidae Arthromacra ﬂavipes Nakane 1 0 Arthromacra takahashi Imasaka 2 1 Oedemeridae Oedemeronia manicata Lewis 0 1 Cantharidae Asiopodabrus kiso kiso Nakane 0 1 Asiopodabrus malthinoides Kiesenwetter 0 1 Asiopodabrus sp. 1 0 Malthodes yukihikoi Takahashi 0 1 Cerambycidae Dinoptera minuta Gebler 3 0 Gaurotes doris Bates 1 0 Parastrangalis nymphula Bates 3 0 Pidonia signifera Bates 5 0 Strangalomorpha tenuis tenuis Solsky 1 0 Chrysomelidae Aphthona perminuta Baly 3 0 Arthrotus niger Motschulsky 4 1 Nonarthra cyanea Baly 1 0 Stenoluperus nipponensis Laboissiere 0 2 Attelabidae Byctiscus venustus Pascoe 0 1 Euops punctatostriatus Motschulsky 1 0 Curculionidae Macrotelephae ichihashii Morimoto 0 1 Mecysolobus erro Pascoe 2 0 Phyllobius annectens Sharp 4 0 Diptera Chironomidae Chironomidae sp. 0 1 Scatopsidae Scatopsidae sp. 1 0 Sciaridae Sciaridae sp. 5 3 Empididae Empididae sp. n. 1 1 2 Empididae sp. n. 2 1 2 Empididae sp. n. 3 1 0 Empididae sp. n. 4 0 2 Empididae sp. n. 5 0 2 Empididae sp. n. 6 0 1 Empididae sp. n. 7 0 1 Dolichopodidae Dolichopodidae sp. 1 0 Phoridae Phoridae sp. 0 2 Syrphidae Allobaccha apicalis Loew 0 1 Betasyrphus serarius Wiedemann 1 0 Brachypalpoides ﬂavifacies Shiraki 1 0 Chalcosyrphus frontalis Shiraki et Edashige 3 1 Cheilosia sp.n.1 1 0 Cheilosia sp.n.2 1 0 Episyrphus balteatus de Geer 0 1 Eristalis cerealis Fabricius 3 0 Eristalomyia tenax Linnaeus 1 0 Graptomyza itoi Shiraki 4 10 Helophilus virgatus Coquillett 3 0 Scaeva komabensis Matsumura 0 1 Takaomyia sexmaculata Matsumura 1 0 Conopidae Conopidae sp. n. 1 2 0 Conopidae sp. n. 2 1 0 Sepsidae Sepsidae sp. 1 1 Chamaemyiidae Chamaemyiidae sp. 1 0

Table 1 Continued

Class Order Family Species Male Female

Lauxaniidae Lauxaniidae sp. 0 1 Psilidae Psilidae sp. 1 0 Heleomyzidae Heleomyzidae sp. 0 1 Chloropidae Chloropidae sp. n. 1 3 4 Chloropidae sp. n. 2 3 2 Chloropidae sp. n. 3 2 0 Chloropidae sp. n. 4 1 0 Anthomyiidae Anthomyiidae sp. n. 1 1 0 Anthomyiidae sp. n. 2 0 1 Calliphoridae Stomorhina obsolete Wiedemann 1 0 Hymenoptera Eulophidae Eulophidae sp. n. 1 1 0 Eulophidae sp. n. 2 1 0 Eulophidae sp. n. 3 0 1 Eupelmidae Eupelmidae sp. n. 1 0 1 Pteromalidae Pteromalidae sp. 1 0 Braconidae Braconidae sp. n. 1 1 0 Braconidae sp. n. 2 0 1 Braconidae sp. n. 3 0 1 Braconidae sp. n. 4 0 1 Braconidae sp. n. 5 0 1 Ichneumonidae Ichneumonidae sp. n. 1 3 0 Ichneumonidae sp. n. 2 1 0 Ichneumonidae sp. n. 3 1 0 Formicidae Camponotus japonicus Mayr 1 0 Formica japonica Motschulsky 1 0 Lasius niger Linnaeus 1 0 Lasius productus Wilson 1 1 Lasius sp. 0 1 Sphecidae Carinostigmus sp. 0 2 Crossocerus sp. 0 1 Psen sp. 4 1 Psenulus sp. 3 2 Rhopalum sp. 0 1 Stigmus sp. 0 1 Colletidae Hylaeus submonticola Ikudome 1 0 Hylaeus thoracicus Ikudome 4 0 Halictidae Lasioglossum afﬁne Smith 36 5 Lasioglossum apristum Vachal 2 0 Lasioglossum exiliceps Vachal 29 17 Lasioglossum laeviventre Perez 12 5 Lasioglossum proximatum Smith 1 1 Lasioglossum sibiriacum Bluthgen 4 0 Lasioglossum transpositum Cockerell 6 6 Lasioglossum sp.n.1 1 0 Lasioglossum sp.n.2 1 0 Andrenidae Andrena dentate Smith 9 1 Andrena hikosana Hirashima 0 1 Andrena parathoracica Hirashima 0 1 Andrena pruniphora Hirashima 29 2 Andrena tsukubana Hirashima 2 0 Andrena yamato Tadauchi et Hirashima 1 0 Apidae Apis cerana japonica Radoszkowski 21 4 Bombus ardens ardens Smith 13 6 Ceratina japonica Cockerell 0 1 Ceratina megastigmata Yasumatsu et 412 Hirashima

female and male flowers, respectively, and among these, orders (P < 0.001; Fig. 1). The visitation frequency of 26 species (21.0% of all collected species) were captured Hymenoptera was significantly greater than the on both flower types (Table 2). The mean (Ϯ standard frequency of Coleoptera, Diptera and Hemiptera error [SE]) visitation frequency per 15 min for all species (Coleoptera vs Diptera, P = 0.999; Coleoptera vs Hemi- was 4.320 Ϯ 0.390 (Fig. 1). There were significant differ- ptera, P = 0.863; Coleoptera vs Hymenoptera, P < 0.001; ences in visitation frequency per 15 min among the insect Diptera vs Hemiptera, P = 0.798; Diptera vs Hymenoptera,

Table 2 Number of species visiting female and male Rhus tri- Lasioglossum exiliceps (P = 0.052; Fig. 3). No signiﬁcantly chocarpa at the Asiu Forest Research Station, Kyoto, in June 2005 female-biased visitation frequency was found for any species. CLASS Order Male Female Total

ARACHNOIDEA Araneae 0 1 1 Potential pollinator introduction INSECTA Hemiptera 4 3 7 Coleoptera 22 15 32 All 15 inflorescences enclosed in mesh bags with bees set Diptera 27 20 39 fruits, whereas 13 of 13 inflorescences bagged without Hymenoptera 31 27 45 bees did not set fruit (Table 3). The fruit sets of inflores- Total 84 66 124 cences bagged with bees varied from 0.07 to 0.40 (Table 3). The fruit sets of inflorescences bagged with a single bee species (Andrena pruniphora, Lasioglossum affine, L. exiliceps P < 0.001; Hemiptera vs Hymenoptera, P < 0.001; Fig. 1). or L. laeviventre Perez) were significantly higher than The number of visitors per 15-min interval did not differ those of control inflorescences, although the fruit set significantly among sampling hours (P > 0.999). varied greatly (from 0.07 to 0.40). In the Hymenoptera, there was a significant difference in average visitation frequency among species (P < 0.001; Discussion Fig. 2); the visitation frequency of Lasioglossum exiliceps Vachal (Halictidae) was significantly higher than the fre- The fact that in total 123 species of insects and one spider quencies of Psenulus sp., Psen sp., Lasioglossum proximatum species belonging to five orders and 46 families visited R. Smith (Halictidae) and Lasius productus Wilson (Formi- trichocarpa (Tables 1,2) indicates that the potential pollina- cidae) (Psenulus sp., P < 0.001; Psen sp., P < 0.001; L. proxi- tors of R. trichocarpa are very diverse. In contrast, the fruit matum, P < 0.001; L. productus, P < 0.001; Fig. 2). In set per inflorescence was 0.388 in 2005. The average fruit contrast, visitation frequencies were not significantly set of several dioecious Anacardiaceae species is 0.32, different among species of Coleoptera or Diptera ranging from 0.01 to 0.80 (Crane & Nelson 1971; Young (Coleoptera, P = 0.701; Diptera, P = 0.054; Fig. 2). 1972; Bawa 1974; Grundwag 1975; Free & Williams 1976). These studies indicate that the fruit set of R. trichocarpa is not low for Anacardiaceae, although there are large Differences in visitation frequency by plant sex species-specific, temporal and spatial variations in fruit set The numbers of flowers per plant were in this family. How does the diverse pollination system 77 254.4 Ϯ 34 984.6 and 31 807.4 Ϯ 10 252.0 (mean Ϯ SE) contribute to the reproductive success of R. trichocarpa? for male and female plants, respectively (P < 0.001). The There was a significant difference in visitation frequen- number of reproductive current-year shoots per plant, cies among insect orders; Hymenopterans were more inflorescences per shoot and flowers per inflorescence for frequent visitors than Coleopterans, Dipterans or female and male plants were 13.8 Ϯ 3.9 and 19.4 Ϯ 4.5, Hemipterans (Fig. 1). Furthermore, visitation frequencies 7.4 Ϯ 0.6 and 9.1 Ϯ 0.4, and 319.2 Ϯ 44.0 and 328.4 Ϯ 60.9, of L. exiliceps were significantly higher than those of some respectively. The estimated fruit set per inflorescence was Hymenoptera (Fig. 2). The same trends were observed in 0.388 Ϯ 0.030 in 2005. The total average visitation fre- the species of Halictidae (e.g. L. affine and L. laeviventre), quency for all species to male flowers was 2.58-fold higher Andrenidae (Andrena dentate and A. pruniphora) and than the frequency to female flowers (mean Ϯ SE, female, Apidae (Apis cerana, Bombus ardens ardens Smith and Cera- 2.527 Ϯ 0.297; male, 6.511 Ϯ 0.656), a statistically signifi- tina megastigmata Yasumatsu et Hirashima), although the cant difference (P < 0.001). One hundred and seven visi- statistical significances were marginal (Fig. 2). Apis cerana tors were significantly from male-biased species, whereas and B. ardens ardens walk and fly on the surface of inflo- 325 visitors were not from biased species. In the rescences and forage nectar and pollen on male flowers or Coleoptera and Diptera, the visitation frequency of each nectar only on female flowers (S. Matsuyama, unpubl. species was unbiased between female and male flowers data, 2005). Some Andrena species and some Lasioglossum (Fig. 3). In Hymenoptera, male-biased visitation frequen- species, which were not identified to species level in the cies occurred in Andrena dentata Smith (Andrenidae), field, walk and fly on the surface of male inflorescences to Andrena pruniphora Hirashima (Andrenidae), Apis cerana collect pollen, whereas they walk and fly on female and japonica Radoszkowski (Apidae) and Lasioglossum affine male inflorescences, and then put their head into flowers Smith (Halictidae) (P = 0.003; A. pruniphora, P < 0.001; Apis to forage for nectar (S. Matsuyama, unpubl. data, 2005). cerana, P < 0.001; Lassioglossum affine, P < 0.001; Fig. 3). In These observations indicate that, in terms of visitation fre- Hymenoptera, the difference between sexes in visitation quency, the eusocial bees (i.e. A. cerana and B. ardens frequency was marginally significant (P < 0.06) for ardens) do not dominantly visit R. trichocarpa, and other

Visitation frequency per 15 min. 0 123 45 Total average (124)

Araneae (1) a Fig. 1 Visitation frequency per 15 min of all insect species and each order (Araneae, Hemiptera (7) a Coleoptera, Diptera, Hemiptera and Hymenoptera). The numbers in parenthe- Coleoptera (32) a Order ses denote the number of species in each order. Error bars indicate the standard Diptera (39) a error. Different letters indicate signiﬁcant differences at P = 0.05 based on a Kruskal– Hymenoptera (45) b Wallis non-parametric test.

Visitation frequency per 15 min. bees in Halictidae, Andrenidae and Apidae play a more 0 0.1 0.2 0.3 0.4 0.5 important role in the reproductive success of R. trichocarpa. Nevertheless, the roles of other visitors (i.e. Colpodes lampros a coleopteran, dipteran and other hymenopteran species) Ampedus tenuistriatus a may not be negligible because they all have the potential Cephaloon pallenes a to transfer pollen through visits to male and female Arthromacra takahashii a Coleoptera flowers, regardless of visitation frequency (Fig. 3). Arthrotus niger a The frequencies of visits by the eusocial bees A. cerana Sciaridae sp. a and B. ardens ardens indicated that they were not domi- Empididae sp. n. 1 a nant visitors (Fig. 2). However, this observation does not Empididae sp. n. 2 a necessarily mean that the role of eusocial bees in the Chalcosyrphus fruntalis a reproductive success of R. trichocarpa is low; Free and Graptomyza itoi a Williams (1972) indicated that intranidal pollen transfer Diptera Sepsidae sp. a occurs in the eusocial bee Apis mellifera Linn, and this Chloropidae sp. n. 1 a transfer may contribute to outcrossing. Hence, eusocial Chloropidae sp. n. 2 a bees may contribute to the reproductive success of R. trichocarpa more than that indicated by the visitation fre- Lasius productus a quency of the bees. Psen sp. a Overall, visitation frequency was biased toward males. Psenulus sp. a However, significantly male-biased visits were observed Lasioglossum affine ab in Andrena dentata, Andrena pruniphora, Apis cerana and L. exiliceps b Lasioglossum affine, but not in the other Hymenoptera, L. laeviventre ab Coleoptera or Diptera (Fig. 3), which may be considered L. proximatum a unbiased and/or occasional visitors. In entomophilous L. transpositum ab species, visits by potential pollinators generally depend

Hymenoptera Andrena dentata ab on the floral rewards of the plant (e.g. Huang et al. 2006). Andrena pruniphora ab We showed that the number of flowers per male R. tri- Apis cerana ab chocarpa plant was 2.43-fold larger than the number of Bombus ardens ardens ab flowers per female plant. In addition, male flowers of this Ceratina megastigmata ab species produce both pollen and nectar, whereas female flowers produce only nectar for animal visitors (S. Mat- Fig. 2 Visitation frequency per 15 min by each visitor species suyama, unpubl. data, 2005). These results infer that the observed on both female and male plants. Error bars indicate the male-biased visitation frequency in R. trichocarpa reflects a standard error. Different letters indicate significant differences correlation between visitation frequency and the amount among the species in each order at P = 0.05 based on a Kruskal– and/or type of floral rewards, and suggest that one highly Wallis non-parametric test. eusocial bee (i.e. A. cerana), one halictid and two andrenid bees have a higher ability to distinguish the floral rewards of R. trichocarpa than other potential pollinators

Visitation frequency per 15 min. 0 0.2 0.4 0.6 0.8 1.0

Colpodes lampros (2) NS Male plants Ampedus tenuistriatus (2) NS Female plants

Cephaloon pallenes (4) NS

Arthromacra takahashii (3) NS Coleoptera Arthrotus niger (5) NS

Sciaridae sp. (8) NS Empididae sp. n. 1 (3) NS

Empididae sp. n. 2 (3) NS

Chalcosyrphus fruntalis (4) NS Graptomyza itoi (14) NS Diptera Sepsidae sp. (2) NS

Chloropidae sp. n. 1 (5) NS

Chloropidae sp. n. 2 (7) NS

Lasius productus (2) NS

Psen sp. (3) NS

Psenulus sp. (5) NS

Lasioglossum affine (41) ***

L. exiliceps (46) NS

L. laeviventre (17) NS

L. proximatum (2) NS

Hymenoptera L. transpositum (12) NS

Andrena dentata (10) **

Andrena pruniphora (31) ***

Apis cerana (25) ***

Bombus ardens ardens (19) NS

Ceratina megastigmata (16) NS

Fig. 3 Visitation frequency per 15 min to female and male ﬂowers by each visitor species. The numbers in parentheses are the numbers of visitors. Error bars indicate the standard error. **P = 0.01 and ***P = 0.001 comparing female and male plants using a Mann–Whitney U-test. NS, not signiﬁcant.

Table 3 Bagged visitors (the number of bagged individuals is in parentheses), Flower Fruits Fruit set Ϯ Ϯ Ϯ number of flowers and fruits per inflores- Bagged visitor (n) Mean SE Mean SE Mean SE cence, and fruit set are shown for each Control (no visitors) 289.2 Ϯ 39.5 (13) 0 Ϯ 0 (13) 0 Ϯ 0(13) bagged inflorescence (the number of Andrena pruniphora (2) 655 78 0.12 samples is in parentheses) Lasioglossum affine (1) 277 20 0.07 Lasioglossum affine (1) 240 83 0.35 Lasioglossum exiliceps (1) 224 23 0.10 Lasioglossum exiliceps (1) 268 108 0.40 Lasioglossum exiliceps (2) 441 172 0.39 Lasioglossum laeviventre (1) 450 116 0.26 Lasioglossum laeviventre (2) 508 146 0.29

SE, standard error.

The fruit sets of the inflorescences bagged with a single (the Ministry of Education, Culture, Sports, Science, and species, that is, Andrena pruniphora, Lasioglossum affine, L. Technology of Japan). exiliceps or L. laeviventre, were significantly higher than the fruit set of the controls (Table 3). Schemske and Horvitz (1984) showed that several visitors with differences in References morphology and taxonomy contributed to the reproduc- Ågren J., Elmqvist T. & Tunlid A. (1986) Pollination by deceit, tive success of Calathea ovandensia. The present study floral sex ratios and seed set in dioecious Rubus chamaemorus showed that the non-social bees A. pruniphora, L. affine, L. L. Oecologia 70: 332–338. exiliceps and L. laeviventre could pollinate female flowers Aigner P. A. (2001) Optimality modeling and fitness trade-offs: of R. trichocarpa (Table 3). Hence, it is suggested that even when should plants become pollinator specialists? Oikos 95: non-social bees, whose visitation frequency varies among 177–184. Bawa K. S. (1974) Breeding systems of tree species of a lowland species, play a role in the reproductive success of R. tropical community. Evolution 28: 85–92. trichocarpa. In short, the pollination syndrome of R. Bierzychudek P. (1987) Pollinators increase the cost of sex by trichocarpa is maintained by a small number of eusocial avoiding female flowers. Ecology 68: 444–447. bees and by a large number of unbiased and infrequent Charlesworth D. (1993) Why are unisexual flowers associated pollinators. with wind pollination and unspecialized pollinators. Ameri- Our results strongly suggest that the pollination system can Naturalist 141: 481–490. of R. trichocarpa includes two types of visitors; the major- Crane J. C. & Nelson M. M. (1971) The unusual mechanism of alternate bearing in the pistachio. HortScience 6: 1022–1035. ity of visitors were non-social bees and other coleopterans Dukas R. (1987) Foraging behavior of 3 bee species in a natural and dipterans with unbiased and/or occasional visits, mimicry system: female flowers which mimic male flowers whereas a minority of visitors were eusocial bees that may in Ecballium elaterium (Cucurbitaceae). Oecologia 74: 256– contribute to the pollination success of R. trichocarpa 263. through male-biased visitation frequency. Elmqvist T., Ågren J. & Tunlid A. (1988) Sexual dimorphism and between-year variation in flowering, fruit-set and pollinator behavior in a boreal willow. Oikos 53: 58–66. Farwig N., Randrianirina E. F., Voigt F. A., Kramer M. & Bohning- Acknowledgments Gaese K. (2004) Pollination ecology of the dioecious tree Com- We owe special thanks to Professor M. Kato, Dr K. miphora guillauminii in Madagascar. Journal of Tropical Ecology 20: 307–316. Yamazaki, Dr S. Imasaka and Dr T. Kishii for classification Free J. B. & Williams I. H. (1972) The transport of pollen on the of insect specimens, and Mr K. Morishita, Mr H. Arai, Mr body hairs of honeybees (Apis mellifera L.) and bumblebees H. Okada and Mr Y. Inoue for invaluable help and advice (Bombus spp. L.). Journal of Applied Ecology 9: 605–615. with fieldwork and laboratory analysis. We appreciate the Free J. B. & Williams I. H. (1976) Insect pollination of Anacardium comments and suggestions by Professor S. Shibata and occidentale L., Mangifera indica L., Blighia sapida Koenig and other members of the Silviculture Laboratory of Kyoto Persea americana Mill. Tropical Agriculture 53: 125–139. University. Finally, we would like to thank the staff of Grundwag M. (1975) Seed set in some Pistacia L. (Anacardiaceae) species after interspecific and intraspecific pollination. Israel Asiu Forest Research Station for permission and support Journal of Botany 24: 205–211. during our field study. This study was supported in part Herrera C. M. (1989) Pollinator abundance, morphology, and by the Center of Excellence for Innovative Food and Envi- flower visitation rate: analysis of the quantity component in a ronmental Studies pioneered by Entomomimetic Sciences plant-pollinator system. Oecologia 80: 241–248.

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