Diverse Visitors with Various Pollinator Importance and Temporal Change in the Important Pollinators of Geranium Thunbergii (Geraniaceae)

Blackwell Science, LtdOxford, UK ERE Ecological Research 0912-38142002 Ecological Society of Japan 173May 2002 488 Important pollinators of Geranium thunbergii I. Kandori 10.1046/j.0912-3814.2002.00488.x Original Article283294BEES SGML

Ecological Research (2002) 17, 283–294

Diverse visitors with various pollinator importance and temporal change in the important pollinators of Geranium thunbergii (Geraniaceae)

IKUO KANDORI* Laboratory of Entomology, Faculty of Agriculture, Kinki University, Naka-machi, Nara, 631-8505, Japan

Assessing pollinator importance of each floral visitor to a plant species is a key to understanding plant–pollinator interaction. The present study examined visitation frequency, pollination efficiency, and pollinator importance of the full range of floral visitors to Geranium thunbergii natural population, by measuring seed-set. During 2 years of observations, the flowers were visited by at least 45 insect species belonging to four orders. Among the main 22 visitor species, 11 species belonging to three orders (Hymenoptera, Diptera, and Lepidoptera) acted as the efficient pollinators. In both years, Hymenoptera, especially bees, was the most important pollinator to G. thunbergii. Thus, the flowers could be considered as bee-pollinated. However, the most important species were not constant between years. The study also documented that the efficient pollinators have larger body sizes. The dish-shaped floral morphology, taxonomically diverse pollinators, and temporal change in the most important pollinators indicate that G. thunbergii–pollinator interaction is a rather generalized system. The results suggest that casual observations of visitation, or even precise measurement of pollinator importance in a single season is insufficient to identify important pollinators.

Key words: generalized system; pollination efﬁciency; seed-set; visit duration; visitation frequency.

INTRODUCTION Pollinator importance can be roughly parti- tioned into two components: ‘quantity’ and ‘qual- Selection for effective pollen transfer and receipt ity’ (Herrera & Jordano 1981; Schemske 1983; has been considered to be the principal force in the Waser & Price 1983). In plant-pollinator systems, evolution of the angiosperm flower. Among ento- the ‘quantity’ component is represented by mophilous (insect-pollinated) plants, some species pollinator abundance or visitation frequency. have evolved highly specialized flowers and are Meanwhile, the ‘quality’ component is mainly pollinated by a single specialist visitor. Most flow- represented by pollination efficiency. There are ers, however, allow access to a variety of visitors largely three ways of measuring the pollination (Baker & Hurd 1968; Faegri & van der Pijl 1979; efficiency. One is measuring pollen loads of forag- Feinsinger 1983). To understand the evolution of ing animals (e.g. Willson et al. 1979; Jennersten floral traits, it is necessary to assess pollinator 1984; Liede 1994). However, this method can be importance (i.e. the relative contributions of the blurred by the fact that some species seldom act different insect visitors to the pollination of a as pollinators although they readily become con- flower). taminated with pollen (Janzen 1977; Harder et al. 1985) and that pollen loads on animals were not strictly correlated with the actual pollination *Author to whom correspondence should be efficiency (Pettersson 1991; Fishbein & Venable addressed. Email: [email protected] 1996). The other two methods reflect pollination Received 28 June 2001. efficiency more reliably. One is measuring pollen Accepted 29 November 2001. removal from the anthers or deposition on the

284 I. Kandori stigma for single visits (e.g. Bertin 1982; Herrera fauna (e.g. Fishbein & Venable 1996; references 1987; Wilson & Thompson 1991; Conner et al. therein). 1995). It has the advantage of assessing pollination efficiency directly. Another is measuring seed-set after a single visit of a given pollinator METHODS (e.g. Motten et al. 1981; Tepedino 1981; Spears 1983). It is an indirect method of assessing polli- Study site and plant species nation efficiency that inevitably includes the effect of the post-pollination process. However, The present study was conducted on a natural using seed-set as a unit of measure has the advan- population of G. thunbergii in the northern suburbs tage of describing the pollination efficiency of a (Kurama) of Kyoto City, Japan (elevation 250 m). flower visitor in terms of the plant’s reproductive The census population occupied an area of success. 10 m × 10 m in fallow land that was located among Recent studies on plant–pollinator interactions cultivated fields along a brook (Kurama River). have shown that, in most cases, flower visitors of The population consisted of well over 100 individ- a certain plant species consist of variety of insects ual plants, although I did not count the number that differ in time and space (e.g. Herrera 1988; precisely, because it was difficult to discriminate 1989; Cane & Payne 1993), and that different the individuals due to the shoots interlacing in the insects have different pollination efficiencies (e.g. dense parts of the population. The study site was Schemske & Horvitz 1984; Herrera 1987; Conner surrounded by forests dominated by Japanese et al. 1995). Nevertheless, few studies have mea- cedars (Cryptomeria japonica) and Japanese cypresses sured both ‘quantity’ and ‘quality’ components for (Chamaecyparis obtusa). Geranium thunbergii plants different visitors to a flower species by using reli- were seen widely around this area, but no other able methods (Young 1988; Pettersson 1991; dense populations were found within a radius of Stone 1996; Gomez & Zamora 1999), or have mea- 200 m. sured pollinator importance relative to other pol- Geranium thunbergii is a perennial wild herb, 30– linators (Schemske & Horvitz 1984; Fishbein & 50 cm high, which is native and distributed all Venable 1996; Olsen 1997). In addition, few over Japan. Usually, it is found on roadsides, grass- hypotheses have been proposed with supporting land, hills and fields. The entire plant is one of the data to explain the difference in pollination effi- most popular folk medicines used as an antidiar- ciency between visitor species, except for morpho- rheal in Japan. Accordingly, most studies on logical difference between insects. G. thunbergii have focussed on its phytochemicals In the present study, I examined visitation and their medicinal use (e.g. Nakanishi et al. frequency, pollination efficiency, and pollinator 1999). The flowers are hermaphroditic, actinomor- importance of the full range of the floral visitors phic, dish shaped, 10–15 mm in diameter, and to Geranium thunbergii Sieb. et Zucc. (Geraniaceae) slightly protandrous (Fig. 1). Usually, G. thunbergii natural population for 2 years by measuring seed- blooms from August to October in central Japan. set per visit on a virgin flower. The primary In this population all the flowers were pink, purposes are to determine which visitors are although white flowers were rarely found around the efficient and/or important pollinators for this area. The sexual parts, consisting of a pistil G. thunbergii, and whether the most important pol- and surrounding five stamens, stand straight in the linators are constant from year to year. In addition, center of the flower. Nectar is secreted at the base I propose body size of floral visitors as one of the of the sexual parts and is easily accessible in the factors affecting pollination efficiency, which is open flower. The petals are tightly furled at night analyzed below. To my knowledge, no other stud- and during rainy weather, when it is difficult for ies have measured pollinator importance within flower visitors to access the nectaries. After polli- the full range of the floral visitors in a natural nation is over, a flower turns into a capsule that community by using seed-set, and in multiple sea- contains five seeds at most. It takes almost 1 sons that may differ in composition of the visitor month for seeds to mature. Geranium thunbergii was

Important pollinators of Geranium thunbergii 285

capsule development was checked and seeds were counted. Seeds that were not developed at that time a were not counted. The number of flowers used for ‘cross-pollinated by hand’, ‘self-pollinated by hand’, ‘unpollinated’, ‘emasculated and unpollinated’, and ‘naturally pollinated’ treatments were 22, 44, 26, 39, and 22, respectively. The number of plants used for each treatment was not counted; however, I chose flowers widely within the population to test as many plants as possible. To assess the flowering phenology of G. thunbergii, the number of all flowers in the study population was counted every 1 or 2 census days b in 1998 and 1999. Nectar sugar concentrations were also measured for haphazardly chosen flowers (n = 18) using hand-held light refractometers (BS- R70; Bellingham & Stanley Ltd, Tunbridge Wells, Kent, UK) that read directly in weight percent (g sugar 100 g solution−1) at approximately noon on 20 September 1998 during fine weather.

Pollination by insects

Fig. 1. (a) Flowers of Geranium thunbergii. (b) Large To measure visitation frequency, pollination effi- Lasioglossum sp. visiting a flower of G. thunbergii. ciency, and pollinator importance of flower visitors to G. thunbergii, field observations were conducted 2 or 3 days per week, from early September to late the dominant flowering species in the study site October in 1998 and from late August to late during observation periods. October in 1999. The observation periods fully covered the peak blooming of the G. thunbergii Floral biology study population for both years. During these observations, virgin first-day flow- To test the reproductive traits of G. thunbergii, floral ers were exposed to a single insect visit, and seed- persistence and seed-set in response to five breeding set was counted once the capsule developed. That treatments were checked during the peak blooming is, 1 or 2 days before the day of observation, each in 1998; virgin (unpollinated) flowers were either bud (approximately 100 buds for one census day) ‘cross-pollinated by hand’ with flowers at least 5 m was bagged with fine-mesh nylon net to exclude away, ‘self-pollinated by hand’, ‘unpollinated’, all visitors, as mentioned above. On the day of ‘emasculated and unpollinated’ where emascula- observation, virgin first-day flowers were uncov- tion was done by tweezers before dehiscence of ered and observed until visited by an insect. To anther, or ‘naturally pollinated’ with unlimited facilitate observation, two to five neighboring vir- insect access. Hand pollination or emasculation was gin flowers were observed at a time. I stayed more done in the morning of the first day of flowering. than 2 m from the target flowers to avoid disturb- In all the treatments except ‘naturally pollinated’, ing the visitors’ foraging behavior. When an insect each flower was kept bagged with 0.25 mm mesh landed on a flower, the following data were nylon net without any support, to exclude all vis- recorded: the species of insect (if immediately itors during flowering. Floral persistence was identifiable), the time and the date, and the dura- counted as the days from the opening to the falling tion of the visit. These ‘single-visit’ flowers were all of the petals. About 10 days after flowering, then immediately emasculated by tweezers, tag-

286 I. Kandori ging the stem right below the flower, and bagged were not used for calculating pollination efficiency again to exclude all visitors thereafter. By emascu- and pollinator importance, and the latter were not lating, seed-set by autonomous-self pollination used for calculating visit duration. The datasets could be avoided, which ensured that all seeds set lacking information about both seed-set and visit in these flowers were pollinated by single visitors. duration were used only for determining visitation About 10 days after a single visit to a flower, frequency. capsule development was checked and seeds counted, as mentioned above. RESULTS At the beginning of the study in 1998, I collected all possible visitors to G. thunbergii for iden- Floral biology tification. Each new species appearing thereafter was also collected and identified. In addition, the The results of floral persistence and seed-set in body length (from the head to the end of the response to five breeding treatments are shown in abdomen) of a subsample of each species was mea- Table 1. The ranking of mean seeds set per flower sured using a magnifying glass. was ‘cross-pollinated’ > ‘naturally pollinated’ > In the field observations, particular care was ‘self-pollinated’ > ‘unpollinated’ > ‘emasculated taken to record flower visitors at the species level, and unpollinated’. ‘Emasculated and unpollinated’ especially for frequent visitors. This was not always flowers had negligible seed-set, which means that possible, however, as very similar congeneric spe- G. thunbergii flowers are not agamospermous. ‘Self- cies could not be reliably separated in the field (e.g. pollinated’ flowers produced seeds, but signifi- species in the genera Lasioglossum, Ceratina, cantly fewer than ‘cross-pollinated’ ones, which Hylaeus). means that the flowers are partially self- In each year, observations were staggered from compatible. ‘Unpollinated’ flowers produced a 9.00 h to 15.00 h, generally for about 4–6 h per few seeds, which was not significantly different day when visitors were active, and in a variety of from negligible seeds produced by ‘emasculated weather conditions, from sunny, hot, still, and dry, and unpollinated’ flowers. This means that to cloudy, cool, breezy, and humid. In a typical day G. thunbergii flowers can produce a small propor- of observation, between 20 and 40 ‘single-visit’ tion of seeds by autonomous-self-pollination. flowers were obtained, although this number var- However, this mechanism is not complete because ied greatly with seasons and weather conditions ‘unpollinated’ flowers produced significantly (minimum one and maximum 64). No observa- fewer seeds than did ‘self-pollinated’ flowers. Usu- tions were made at night and during rain, as visi- ally, autonomous-self-pollination occurred after tors to G. thunbergii were very uncommon at that 15.00 h on the unpollinated flowers in the first day time. Similarly, no observations were made before of blooming, when a pistil gradually dehisced, 9.00 h or after 15.00 h, because visitors were rare expanded, and touched surrounding stamens. at that time. Floral persistence was negatively correlated with Visitation frequency was obtained as the num- the number of seeds set (Table 1). All ‘cross- ber of ‘single-visit’ flowers for each insect species pollinated’ and ‘naturally pollinated’ flowers, during the observations. Pollinator importance which had approximately maximum seed-set, was calculated as the overall seeds set by a given lasted 1 day. On the contrary, ‘emasculated and visitor relative to the overall seeds set by all the unpollinated’ flowers, which had no opportunities visitors during the observations. to pollinate, lasted 2.58 days. This means that The ideal data set for a single visit on a flower G. thunbergii flowers extend their floral persistence included information about the visitor species, the if they are not pollinated. time and the date, the visit duration, and the The flowering phenology of the G. thunbergii number of seeds set. However, some data sets were study population is shown in Fig. 2. Flowers began missing information regarding either seed-set or to bloom in early August in 1999, full-bloomed visit duration for several reasons, such as seed pre- in late September and finished blooming in early dation by herbivores before counting seed-set. November in both 1998 and 1999. In 1999, Regarding the data analysis, the former data sets flower density was higher throughout the season,

Important pollinators of Geranium thunbergii 287

Table 1 Floral persistence (days) and seed-set per ﬂower of Geranium thunbergii in response to ﬁve breeding test treatments

d.f. MS FP

(a) Results of ANOVA Floral persistence Treatment 4 13.90 46.18 <0.0001 Error 128 0.30 Seeds set/ﬂower Treatment 4 104.37 40.74 <0.0001 Error 148 2.56 (b) Mean ± SE (n) of each treatment Treatment Floral persistence Seeds set/ﬂower Cross-pollinated by hand 1.00 ± 0.00a (20) 4.46 ± 0.31a (22) Self-pollinated by hand 1.27 ± 0.11a (34) 2.84 ± 0.32b (44) Unpollinated 2.08 ± 0.16b (24) 0.92 ± 0.36c (26) Emasculated, unpollinated 2.58 ± 0.11c (33) 0.03 ± 0.03c (39) Naturally pollinated 1.00 ± 0.00a (22) 3.86 ± 0.34ab (22)

Figures in the same column sharing a superscript do not differ signiﬁcantly (P > 0.05 Tukey’s HSD test).

Visitation frequency During 2 years of observations, the flowers were visited by at least 45 insect species, including bees, which were the most diverse group (20 species), wasps (two species), ants (four species), hoverflies (seven species), butterflies (seven species), sphinx moths (one species), and thrips (one species), belonging to four orders, with Hymenoptera being the most diverse order (26 species), and with Diptera (10 species) and Lepidoptera (eight species) accounting for most of the rest (Table 2). Fig. 2. Flowering phenology of Geranium thunbergii. Relative abundance of the three main orders Numbers of flowers in 10 m × 10 m of each 1998 and varied significantly between years (χ2 43.61, 1999 study population are shown. = d.f. = 2, P < 0.0001; Table 2). In each year, Hymenoptera was the most abundant (63.1% and and the peak density was more than twofold 75.6% of the total number of visits in 1998 and higher compared with 1998. 1999, respectively). However, Lepidoptera was Nectar concentration averaged 69.64 ± 1.35% more abundant in 1998 (19.6%) compared with SE (n = 18) on a weight-of-solute/weight-of- 1999 (6.4%). As a group, bees were the most solution basis. abundant in each year (50.8% and 60.4% in 1998 and 1999, respectively). At the species level, in 1998, Zizeeria maha was the most abundant Pollination by insects (13.3%), followed by large Lasioglossum spp. A total of 1156 visits to G. thunbergii virgin flowers (11.3%) and Ceratina spp. (10.0%), while in 1999, (301 and 855 visits in 1998 and 1999, respec- Apis cerana was the most abundant (22.3%), tively) for a total of 42 census days (14 and 28 days followed by Megachile tsurugensis (10.2%) and in 1998 and 1999, respectively) were observed. Paratrechina sakurae (9.4%). 288 I. Kandori ) n SE ( ± 131.0 (2) 9.5 (2) 6.1 (8) 5.6 (24) 4.4 (16) 2.2 (14) 1.7 (39) 1.6 (34) 2.6 (40) 10.3 (4) 1.7 (48) 1.4 (9) 0.6 (74) 0.2 (194) 0.4 (110) 34.2 (8) 1.0 (92) 0.6 (13) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Mean Geranium thunbergii isitors to (%) duration (s) Visit Importance set 00 40 0 0.0 170 0.5 0.0 0.0 3.1 17 0.0 2.0 29.4 13.1 0 0.0 2.0 74 11.5 6 0.0 0 8.85 8.1 1.80 3 0.0 12 13.9 1.5 0.0 0.4 178.8 1.4 – 10 33847 3.0 85 40.0 14.1 4.1 10.157 12.4 51 9 13623 17.1 15.3 16.1 1.1 9 10.6 14 5.5 6.9 15 1.1 4.2 25.7 1.8 17.6 Overall seeds 1 ** ** ns ** ns ** ** ** ** * ns ns ns ns ) n 0.05 by using the sequential Bonferroni technique). Only species SE ( =

± α 0.49 (14) 0.14 (195) 0.18 (74) 0.13 (24) 0.18 (48) 0.46 (14) 0.00 (2) 0 10 0.0 1.2 25.5 0.37 (32) 0.24 (13) 0.20 (110) 0.76 (9) 0 210.63 (8) 0.0 2.5 5 3.9 0 1.5 0.0 14.4 0.05 (80) 0.22 (85) 0.00 (22) 0.28 (37) 0.15 (33) 0.25 (4) 0 1 0.0 0.1 30.8 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Mean Visitation frequencyVisitation Seed-set per visit . 1998 (%) 1999 (%) 1998 1999 1998 1999 0.01 (adjusted the probabilities of error to < P . 8.85.0 34 (11.3) 14 (4.7) 58 (6.8) 10 (1.2) 1.55 0.00 6.85.8 30 10.0 10 (1.2) 25 (8.3) 0.86 15 (1.8) 0.18 Hylaeus floralis sp. 18. (mm) . 0.05; **, and Body length < P 3 2 Lasioglossum Lasioglossum occidens 4 0.07; *, and < Ceratina japonica and P 5 < Hylaeus matsumurai and , (Halictidae) 7.2 3 (1.0) 13 (1.5) 1.21 (Megachilidae) 9.4 24 (8.0) 87 (10.2) 1.70 (Formicidae) 1.8 0 (0.0) 80 (9.4) 0.15 (Megachilidae) 8.4 0 (0.0) 74 (8.7) 1.00 (Megachilidae) 10.5 18 (6.0) 16 (1.9) 2.06 spp. (Halictidae) spp. (Halictidae) (Megachilidae) 15.3 0 (0.0) 2 (0.2) 5.00 -test against control (‘emasculated and unpollinated’ treatment in Table-test against control (‘emasculated and unpollinated’ treatment in 1). (Eumenidae) 10.0 1 (0.3) 7 (0.8) 0.63 (Megachilidae) 11.9 0 (0.0) 14 (1.6) 1.21 (Halictidae) 5.8 1 (0.3) 0 (0.0) 3.00 (1) 3 0 0.9 0.0 15.0 (1) (Megachilidae) 12.8 0 (0.0) 9 (1.1) 2.33 (Formicidae) 4.5 2 (0.7) 0 (0.0) 0.00 (2) 0 0 0.0 0.0 481.0 U (Vespidae) 16.2 22 (7.3) 26 (3.0) 0.60 (Anthophoridae) 4.3 0 (0.0) 4 (0.5) 0.25 (Apidae) 12.0 0 (0.0) 13 (1.5) 0.31 (Formicidae) 3.5 12 (4.0) 16 (1.9) 0.33 (Apidae) 10.5 4 (1.3) 191 (22.3) 1.78 Lasioglossum mutilum Lasioglossum japonicum Ceratina flavipes Hylaeus nippon spp. (Anthophoridae) Body length, visitation frequency, seed-set per visit, overall seeds set, pollinator importance, and visit duration of insect v Body length, visitation frequency, spp. (Colletidae) Lasioglossum Lasioglossum Non-significant; (*), 0.05 Mann–Whitney Includes Includes Includes Includes Coelioxys yanonis Apis cerana Chalicodoma spissula Lasius niger Paratrechina sakurae Formica japonica Large Large Lasioglossum scitulum Halictus aerarius Coelioxys fenestrata Coelioxys acuminata Ceratina Polistes chinensis Bombus diversus Small Megachile tsurugensis Megachile remota Ceratina iwatai Hylaeus Eumenes samuray Formicidae gen. sp. – 0 (0.0) 1 (0.1) 0.00 (1) 0 0 0.0 0.0 – ns 1 2 3 4 5 Hymenoptera with at least 10 samples are tested. Table 2 Visitor Important pollinators of Geranium thunbergii 289 ) n SE ( ± 11.4 (62) 1.8 (1034) 10.9 (7) 7.5 (113) 35.6 (5) 8.4 (3) 5.8 (189) 28.5 (4) 5.9 (6) 3.2 (28) 3.0 (26) 1.3 (732) 13.3 (70) 25.2 (14) 11.8 (13) 6.1 (56) 0.1 (3) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Mean (%) duration (s) Visit Importance set 1 4 0.3 0.5 22.1 5 90 1.5 1.1 0 19.3 0.0 0.0 – 79 24 0 2.1 2.7 2.8 69.8 0.0 88.7 16 525 4.8 16 0.6 7.5 37.7 1.9 106.5 23 16 6.9 1.9 57.9 Overall seeds 1 * ns (*) ns ** ns ns ns ) n 0.05 by using the sequential Bonferroni technique). Only species SE ( =

± α 0.55 (13) 0.00 (17)0.05 (1122) 334 0 844 100 0 100 0.0 27.9 0.0 – 0.72 (7) 13 1 3.9 0.1 36.1 0.00 (17) 0.14 (113) 47 36 14.1 4.3 71.7 0.12 (26) 0.23 (26) 1.67 (3) 0 10 0.0 1.20.33 (3) 32.7 0 1 0.0 0.1 0.2 0.00 (5) 0 0 0.0 0.0 63.2 0.10 (184) 66 57 19.8 6.8 58.1 0.35 (13) 0.87 (4) 8 2 2.4 0.2 52.0 0.82 (6) 1 5 0.3 0.6 68.5 0.18 (62) 0.17 (56) 0.06 (808) 221 751 66.2 89.0 13.4 0.14 (68) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Mean Visitation frequencyVisitation Seed-set per visit . 1998 (%) 1999 (%) 1998 1999 1998 1999 0.01 (adjusted the probabilities of error to < P . Hylaeus floralis sp. 18. (mm) . 0.05; **, and Body length < P Lasioglossum Lasioglossum occidens 0.07; *, and < Ceratina japonica and P < (Syrphidae) 6.0 10 (3.3) 60 (7.0) 0.46 Hylaeus matsumurai and , (Phasiidae) 6.9 0 (0.0) 3 (0.4) 3.33 (Syrphidae) 4.0 4 (1.3) 24 (2.8) 0.19 (Syrphidae) 9.9 2 (0.7) 2 (0.2) 2.50 (Syrphidae) 9.9 10 (3.3) 4 (0.5) 0.69 (Syrphidae) 8.5 19 (6.3) 37 (4.3) 0.70 (Calliphoridae) 4.4 1 (0.3) 4 (0.5) 0.00 -test against control (‘emasculated and unpollinated’ treatment in Table-test against control (‘emasculated and unpollinated’ treatment in 1). (Hesperiidae) 16.0 0 (0.0) 1 (0.1) 0.00 (1) 0 0 0.0 0.0 40.0 (1) (Lycaenidae) 12.0 3 (1.0) 3 (0.4) 1.00 U (Sphingidae) 27.3 0 (0.0) 3 (0.4) 0.33 (Hesperiidae) 15.0 8 (2.7) 18 (2.1) 0.54 (Lycaenidae) 9.0 40 (13.3) 23 (2.7) 0.66 (Pieridae) 14.5 1 (0.3) 0 (0.0) 0.00 (1) 0 0 0.0 0.0 44.0 (1) (Pieridae) 19.4 0 (0.0) 1 (0.1) 0.00 (1) 0 0 0.0 0.0 9.0 (1) (Pieridae) 17.2 7 (2.3) 6 (0.7) 1.62 Lasioglossum mutilum Lasioglossum japonicum Ceratina flavipes Hylaeus nippon sp. (Syrphidae) – 0 (0.0) 1 (0.1) 0.00 (1) 0 0 0.0 0.0 90.0 (1) Continued Non-significant; (*), 0.05 Mann–Whitney Includes Includes Includes Includes Thysanoptera 0 (0.0) 17 (2.0) 0.00 Pieris rapae Gymnosoma rotundata Diptera gen. spp. – 5 (1.7) 2 (0.2)Lepidoptera 2.00 59 (19.6) 55 (6.4) 0.73 Thysanoptera gen. sp. 1.0 0 (0.0) 17 (2.0) 0.00 Paragus haemorrhous Parnara guttata Cephonodes hylas Paragus Syrphidae gen. sp.Stomorhina obsoleta –Pelopidas mathias 1 (0.3) 0 (0.0) 5.00 (1)Diptera 5 0 1.5 0.0 36.0 (1) 52 (17.3) 137 (16.0) 0.67 Episyrphus balteatus Metasyrphus ferquens Pieris melete Zizeeria maha Lampides boeticus Eurema hecabe Sphaerophoria macrogaster Betasyrphus serarius Hymenoptera 190 (63.1) 646 (75.6) 1.20 ns 1 2 3 4 5 Visitor Lepidoptera Total 301 (100) 855 (100) 1.05 Thysanoptera with at least 10 samples are tested. Table 2 Table Diptera Subtotals 290 I. Kandori

Table 3 ANCOVA of seed-set per visit by four groups of insects with their body length as covariate*

Source d.f. MS FP

Insect group1 3 0.09 0.36 0.784 Body length 1 1.87 7.29 0.015 Interaction 3 0.37 1.43 0.269 Error 17 0.26

*Only the species that recorded at least ﬁve visits are analyzed. 1Four groups with sample sizes (number of species con- cerned) are as follows: bees (12), other Hymenoptera (4), Diptera (5), and Lepidoptera (4). Thysanoptera was excluded because of its small sample sizes.

Fig. 3. Relation between body length and mean seed-set per visit for each insect species. Only species Pollination efficiency with at least five samples in the 2-year total are shown (Table 2). (), Hymenoptera; (), Diptera; As a measure of pollination efficiency, seed-set per (), Lepidoptera; (), Thysanoptera. Regression: visit was calculated for each visitor species y = 0.065 + 0.088x, r2 = 0.308, n = 26, F = 10.67, (Table 2). Among the main 22 visitor species P = 0.003. (those recording at least 10 visits in the 2-year total), 20 species had a measurable seed-set. Com- parisons with control flowers (‘emasculated and (Table 2). At the order level, Hymenoptera was the unpollinated’ treatment in Table 1) were statisti- most important in each year (66.2% and 89.0% of cally significant for 11 of these species (eight spe- importance in 1998 and 1999, respectively), cies in Hymenoptera, two in Diptera, and one in although both Diptera and Lepidoptera were more Lepidoptera), thus indicating that they were effi- important in 1998 compared with 1999 (19.8% cient pollinators of G. thunbergii. The results indi- for Diptera and 14.1% for Lepidoptera in 1998, cate that the efficient pollinators consisted of and 6.8% and 4.3%, respectively, in 1999). Rela- diverse insects belonging to three orders. tive pollinator importance of the main three orders ANCOVA of seed-set per visit was calculated for changed significantly between years (χ2 = 80.53, the main groups of insects with their body length d.f. = 2, P < 0.0001; Fig. 4a). As a group, bees were as the covariate (Table 3). This revealed that body the most important in each year (59.0% and length significantly affected seed-set per visit, 85.4% in 1998 and 1999, repectively). At the whereas insect group did not. All of the main species level, in 1998, Coelioxys acuminata (17.1%), insect species were then combined to analyze linear M. tsurugensis (15.3%), and large Lasioglossum spp. regression (Fig. 3). This revealed that the average (14.1%) were equally the most important. While seed-set per visit correlated significantly with body in 1999, A. cerana alone was the most important length, indicating that larger insects were more (40.0%): it contributed to seed-set over twofold efficient pollinators to G. thunbergii. more than the second best species, M. tsurugensis (16.1%). Relative pollinator importance of the best 10 important pollinator species changed sig- Overall seeds set and pollinator importance. nificantly between years (χ2 = 358.65, d.f. = 9, During the 2 years of observations, a total 1178 P < 0.0001; Fig. 4b). seeds were produced on ‘single-visit’ flowers by The results indicate that Hymenoptera, espe- insect visitors (334 and 844 seeds in 1998 and cially bees, was constantly the most important pol- 1999, respectively; Table 2). Pollinator importance linator to G. thunbergii. However, at the species was calculated as overall seeds set of a given visitor level, the most important pollinators changed divided by those of all visitors for each year between years. Important pollinators of Geranium thunbergii 291

Visit duration cantly shorter than that of Diptera or Lepidoptera, whereas there was no significant difference Mean visit duration of ants was the longest of all between Diptera and Lepidoptera (Table 4). visitor species measured (Table 2). Except for ants, The average duration of a visit tended to corre- mean visit duration of Hymenoptera was signifi- late negatively with body length within bees, Hymenoptera, Lepidoptera, or all the main three orders combined, but the significance was detected only within bees (regression: y = 34.88 − 2.54x, r2 = 0.603, n = 12, F = 15.20, P = 0.003; when species recording at least five visits in the 2-year total were considered).

DISCUSSION

The results of the breeding test revealed some reproductive traits of G. thunbergii (Table 1). It is not agamospermous, meaning that all seeds are produced by pollination. It obtains approximately full seed-set by cross-pollination, which was usually achieved by a day of natural pollination by insects in the study population (Table 1). It is partially self-compatible and produces a few seeds by incomplete autonomous-self pollination when it is not pollinated by pollinators. Geranium thunbergii is also adaptive in that it extends the floral persistence in the case of such pollinator limitation. The extension of the floral persistence has been Fig. 4. Pollinator importance of the main insect taxa reported for other plant species (e.g. Motten in 1998 () and 1999 (). (a) The main three orders are shown. (b) The best 10 species are shown, which 1983). were selected by simply averaging 2 years’ pollinator The present study documented temporal change importance. Species in the figure are arranged in order in the most important pollinator species. It may, of pollinator importance in 1998. in part, be brought about by concentrative visiting

Table 4 Visit duration (s) of the main three insect groups (Hymenoptera without ants, Diptera and Lepidoptera)*

d.f. MS FP

(a) Results of ANOVA Insect group 2 6036 16.01 <0.0001 Error 20 377 (b) Mean ± SE (n) of each insect group Insect groups Visit duration Hymenoptera without ants 12.38 ± 2.09a (14) Diptera 60.34 ± 10.89b (5) Lepidoptera 58.00 ± 19.09b (4)

*In this case, only the species that recorded at least five visits were considered. Ant group was excluded from the analysis because of its small sample size. Figures sharing a superscript do not differ significantly (P > 0.05; Tukey’s HSD). n, number of species. 292 I. Kandori by honeybees in 1999. During that season, flower density was always higher, and the peak density was more than twofold higher compared with 1998 (Fig. 2). Bees are known to forage concentra- tively by communicating in the hive once the flower resources become rewarding. Motten (1983) showed that the abundance of Apis mellifera on Erythronium umbilicatum varied greatly among sites and years. During the observations in the present study, I might have failed to detect a large number of thrips visiting G. thunbergii virgin flowers. They were found on various parts of the flower and often hid among the sexual parts and at the base of petals, remaining in the flower, eating pollen. However, as shown in Table 2, they did not pollinate G. thunbergii at all. Therefore, contamination of thrips to virgin flowers probably had little effect on measuring seed-set. Difference in pollinator importance among pollinator species was due to variation in visitation frequency and pollination efficiency (seed-set per visit). Some visitors were efficient but not frequent pollinators (e.g. Pieris rapae), and others were frequent but not efficient (e.g. Paratrechina sakurae, Hylaeus spp., and small Lasioglossum spp.), resulting in both being less important pollinators (Table 2). The most important pollinators were both efficient and frequent species. Such taxonomic variation in pollinator importance was not unusual in other plant-pollinator systems (e.g. Schemske & Horvitz 1984; Young 1988; Pettersson 1991; Fishbein & Fig. 5. Schematic diagram showing different-sized Venable 1996; Stone 1996; Olsen 1997; Gomez & insects collecting nectar on a Geranium thunbergii. (a) Zamora 1999). However, few studies have evalu- Large insects were usually located upon the sexual parts ated its temporal difference (Fishbein & Venable in the center. (b) Small insects were usually located on 1996; the present study). the peripheral petals, resulting in far fewer contacts The present study has also documented that the with the sexual parts. efficient pollinators have larger body sizes (Fig. 3). The difference in pollination efficiency may be much less contact with the sexual parts (Fig. 5b). partly explained by their differences in foraging Maybe it is difficult for large insects to land on the behavior according to their body sizes (Fig. 5). petals because of their size and weight. Similarly, While collecting nectar from G. thunbergii flowers, it may be difficult for small insects to collect nectar large insects, such as A. cerana, M. tsurugensis, and by positioning themselves on the sexual parts large Lasioglossum spp., were often located upon the because of their short tongues. The positive corre- sexual parts and held them in their legs, thus their lation between pollination efficiency and pollina- legs and body could easily come into contact with tor size has also been reported between honeybees the sexual parts (Figs 1b,5a). In contrast, small or worker bumblebees, and halictid bees visiting insects such as ants, Hylaeus spp., and small Lasi- Campsis radicans (Bertin 1982), and between bum- oglossum spp., were often located on the peripheral blebees and halictids visiting Linaria vulgaris petals, walking on them, and encircling the nec- (Arnold 1982). However, these studies compared taries on the base of the sexual parts, resulting in only a small number of species. Important pollinators of Geranium thunbergii 293

Most theoretical and empiric studies on plant– Thanks are also offered to Y. Sakuratani for his pollinator interactions use specialization as a cen- critical reading of the manuscript. tral idea. Nevertheless, recent studies have pointed out that generalization is at least as frequent as specialization in pollination systems, with increas- ing evidence indicating that most flowering plants REFERENCES are pollinated by moderate to high numbers of insect species, and that most floral visitors usually ARNOLD R. M. (1982) Pollination, predation, and seed set in Linaria vulgaris (Scrophulariaceae). visit many different hosts (Schemske 1983; American Midland Naturalist 107: 360–369. Herrera 1996; Waser et al. 1996; Gomez & BAKER H. G. & HURD P. D. (1968) Intrafloral Zamora 1999). The mutualistic interaction ecology. Annual Review of Entomology 13: 385–414. between G. thunbergii and its pollinators can be BERTIN R. I. (1982) Floral biology, hummingbird considered as a rather generalized system for three pollination and fruit production of trumpet reasons. First, the traits of the flower (open and creeper (Campsis radicans, Bignoniaceae). American dish shaped) allow almost every visitor to obtain Journal of Botany 69: 122–134. rewards and to act as potential pollinators. Second, CANE J. H. & PAYNE J. A. (1993) Regional, the efficient pollinators of this flower were, in fact, annual, and seasonal variation in pollinator guilds: taxonomically diverse insects, belonging to three intrinsic traits of bees (Hymenoptera: Apoidea) orders. Third, the most important pollinator spe- underlie their patterns of abundance at Vaccinium cies changed between years. As has often been ashei (Ericaceae). Annals of the Entomological Society of America 86: 577–588. noted, generalization is favored within the plant CONNER J. K., DAVIS R. & RUSH S. (1995) The population by temporal unpredictability in polli- effect of wild radish floral morphology on pollina- nator assemblage (e.g. Fishbein & Venable 1996; tion efficiency by four taxa of pollinators. Oecologia Herrera 1996; Waser et al. 1996; references 104: 234–245. therein). Thus, temporal change in the most FAEGRI K. & VAN DER PIJL L. (1979) The Prin- important pollinators will make pollinator- ciples of Pollination Ecology, 3rd edn. Pergamon, mediated selective pressure inconsistent, and spe- Oxford. cialization of floral traits towards particular visitor FEINSINGER P. (1983) Coevolution and Pollina- species unlikely. However, some kind of specialization. In: Coevolution (eds D. J. Futuyma & M. tion towards bees may occur because bees were Slatkin) pp. 282–310. Sinauer, Sunderland. constantly the most important pollinator group for FISHBEIN M. & VENABLE D. L. (1996) Diversity G. thunbergii. and temporal change in the effective pollinators of Asclepias tuberosa. Ecology 77: 1061–1073. In conclusion, quantitative analysis by measur- GOMEZ J. M. & ZAMORA R. (1999) Generalization ing seed-set revealed that G. thunbergii was polli- vs. specialization in the pollination system of Hor- nated mainly and constantly by bees. However, at mathophylla spinosa (Cruciferae). Ecology 80: 796– the species level, the most important pollinators 805. were different between the 2 years, and efficient HARDER L. D., THOMSON J. D., CRUZAN B. M. pollinators were not only found within bees but & UNNASCH R. S. (1985) Sexual reproduction also within other taxonomic groups. The results and variation in floral morphology in an ephem- indicate that the mutualistic interaction between eral vernal lily, Erythronium americanum. Oecologia G. thunbergii and its pollinators is a rather gener- 67: 286–291. alized system. It was also found that larger visitors HERRERA C. M. (1987) Components of pollinator were more efficient pollinators of this flower. ‘quality’: comparative analysis of a diverse insect assemblage. Oikos 50: 79–90. HERRERA C. M. (1988) Variation in mutualisms: the spatio-temporal mosaic of a pollinator assem- ACKNOWLEDGEMENTS blage. Biological Journal of the Linnean Society 35: 95–125. Insect identifications were generously provided HERRERA C. M. (1989) Pollinator abundance, mor- by O. Tadauchi (Apoidae), Y. Maeta (Apoidae), phology, and flower visitation rate: analysis of the M. Kato (Apoidae), H. Sakata (Formicidae), T. ‘quantity’ component in a plant-pollinator sys- Kimura (Syrphidae), and H. Oishi (Syrphidae). tem. Oecologia 80: 241–248. 294 I. Kandori

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