Article The Floral Biology,Breeding System and Pollination Efficiency of Schima superba Gardn. et Champ. ()

Hanbo Yang 1,2, Rui Zhang 1,*, Ping Song 1 and Zhichun Zhou 1

1 Research Institute of Subtropical Forestry; Zhejiang Provincial Key Laboratory of Tree Breeding, Fuyang 311400, ; [email protected] (H.Y.); [email protected] (P.S.); [email protected] (Z.Z.) 2 Sichuan Academy of Forestry, Chengdu 610081, China * Correspondence: [email protected]; Tel.: +86-137-0681-6436

Received: 21 July 2017; Accepted: 11 October 2017; Published: 24 October 2017

Abstract: Schima superba Gardn. et Champ. is a perennial, evergreen tree valued for its eco-protection and commercial values in China. In this study, we investigate the breeding system, reproductive ecology and pollination biology of S. superba in a seed orchard. The flowers are hermaphrodite and protogynous. The viability of the pollen is inactivated rapidly, and the stigma maintains a high receptivity within the flower lifespan. Flowers typically offer pollen and nectar to visitors. The flowers possess a typical insect pollination syndrome, and three visitors (Apis cerana cerana Fabricius, Protaetia brevitarsis Lewis, and Popillia mutans Newman) are observed on flowers during the study period. The visitation frequency per minute and capability of pollen removal and deposition of A. cerana are significantly higher than P. brevitarsis and P. mutans, although the pollinator efficiency is lower than those shown by the two beetles. Fruit set (28.27%) and seed set (6.57%) percentages resulting from open-pollination are significantly lower than those resulting from cross-pollination (fruit/seed set, 43.73%/11.66%), and the pollen limitation index (L) was 0.34, suggesting that seed production is pollen-limited in the seed orchard. The pollen/ovule ratio (P/O) and outcrossing index (OCI) values are 6686.67 and 4, respectively. The self-incompatibility index (ISI) was estimated to be 0.95. Results from hand-pollination, pollen tube growth experiments and the ISI value show that S. superba is late-acting self-incompatible. The synthetic results indicate that A. cerana is the most efficient pollinator of S. superba, and seed production is frequently limited by pollinators, fruit abortion, and pollen quality.

Keywords: breeding system; pollination; floral biology; Schima superba Gardn. et Champ.

1. Introduction Breeding systems are important and often neglected aspects of reproductive biology; the main research content includes floral characteristics, pollination and mating systems [1]. The floral biology, pollination pattern, and behavior are closely related to reproductive fitness and are thus subjected to strong selective forces [2]. The reproductive success of is often limited by lack of pollination, mate availability and fecundity and mate limitation [3]. Flower arrangement and reward availability within plants are also important factors that influence mating success [4]. The timing of pollen presentation and stigma receptivity at both the individual and population levels may strongly correlate with mate availability, and species with long flowering periods may avoid pollination limitation and reproductive deficit as a consequence of an increased overlap with insect activity [5]. Individuals with hermaphrodite flowers may resort to self-pollination, which leads to maximum seed setting but minimum genetic variation in their offspring [6]. However, geitonogamy may also mitigate pollen limitation in self-compatible plants [7]. Therefore, floral syndrome and breeding system investigations are not only conducive to understanding the pollination

Forests 2017, 8, 404; doi:10.3390/f8100404 www.mdpi.com/journal/forests Forests 2017, 8, 404 2 of 12 mechanism and mating patterns but also have scientific significance for further investigating the evolution and environmental compatibility of plants. Pollination is a key stage of generative propagation, and the dispersal of plants is closely linked with pollinators. In addition, pollination has important application value for biodiversity. Approximately 65% of flowering plants are insect-pollinated [8]. Insect pollination promotes the rapid development of flowering plants. Pollination mediates male-female interactions and is dependent on abiotic and biotic factors [6]. The pollination success of pollinator-dependent species is highly influenced by the frequency of visits, the intrafloral foraging behavior of pollinators and the breeding system [9]. In self-incompatible plants, the visitation rate and foraging behavior of pollinators within plants influence the amount of self-pollen transferred and may eventually lead to pollen limitation [9]. A lack of faithful pollinators, a few pollinators or low pollination efficiency in insect-pollinated plants can result in low pollination intensity and reduced seed yield, and the resulting flower-visiting behavior would also affect the seed yield and quality [10]. Pollination efficiency is directly related to pollination amount and quality and is affected by flower-visiting behavior [11]. Therefore, the interaction among population structure, pollination behavior, and pollination efficiency is very complex and requires synthetic and meticulous research. Theaceae is a family of subtropical and tropical trees in with approximately 17–20 genera and 500 species [12]. Members of this family are predominantly pollinated by insects [6,13]. Previous studies on the breeding biology of Theaceae have mainly focused on Ternstroemia laevigata [13], Ternstroemia dentate [13], Camellia japonica L. [14], and (DC.) Korth [6]. Schima superba Gardn. et Champ. is the most widely distributed species of Schima plants in China. This species is not only used as a biological firebreak and an ecological protection tool, but it is also considered a valuable timber tree [15]. However, we know little about the reproductive biology of S. superba. Studies examining pollination and breeding biology have great potential for revealing insect/plant interactions and their underlying importance in speciation and diversification, genetic fitness, reproductive efficiency and success in areas of applied forest science [16]. Thus, we analyzed the floral syndrome, breeding system and pollination efficiency of S. superba: (1) What are the characteristics of the breeding system of S. superba? (2) What are the barriers to fruit and seed set in the flowering and pollination processes? (3) What are the effective pollinators of S. superba, and what are their effects on fruit and seed set? The answers to these questions can contribute to artificial pollination, the development of crossbreeding and variety improvement, as well as promote seed production.

2. Materials and Methods

2.1. Study Species and Site Schima superba Garden. et Champ. is a monoecious, large evergreen tree native to China [17]. The leaves are leathery or slightly leathery; the flowers are white and the ovary hairy [17]. Based on long-term observations, the fruit-bearing period of S. superba lasts 1.5 years and seed maturation occurs in approximately October of the following year. The present investigation was conducted in a seed orchard in Jinhua City, eastern China (29◦0801600 N, 119◦2704700 E). The average annual rainfall is 1438.9 mm, the elevation is 42 m, and the mean annual temperature is 17.7 ◦C. The surrounding vegetation consisted mainly of Erigeron annuus (L.) Pers., Setaria viridis (L.) Beauv., Plantago depressa Willd, Cinnamomum camphora (L.) Presl., Liriodendron chinense (Hemsl.) Sarg. and Celtis sinensis Pers., etc. The selected trees were of similar age and were cultivated through cuttings.

2.2. Floral Biology The total number of inflorescences per individual was counted based on 30 randomly selected trees. We recorded the total number of flowers, flowering period, flower lifespan, and timing of anther dehiscence. We measured the length of the stigma and stamen (LS and LOS) and petal and corolla (LP and LC) and calculated the mean values of 60 replicates. All floral measurements were made to 0.01 mm using digital calipers. The number of pollen grains was estimated using a hemocytometer [18], Forests 2017, 8, 404 3 of 12 and ovule numbers were counted directly, based on 60 replicates. Twenty flowers were randomly selected and bagged before anthesis, and the nectar volumes were measured using 20-µL Hirschmann “minicap” calibrated capillary tubes. Stigma receptivity and pollen viability were measured three times a day (at 09:00, 15:00, and 21:00) for a single live flower using a benzidine-H2O2 test [18] and an in vitro culture method, respectively. The flowers used to measure floral structure, pollen grains, nectar volumes, stigma receptivity, and pollen viability were collected separately.

2.3. Breeding System To determine the breeding system, we carried out five pollination treatments on five trees with a large amount of flowers: (1) apomixis: 20 artificial emasculation flowers were bagged without pollination; (2) self-pollination using the same flower (SPSF): 20 flowers were hand-pollinated with the pollen from the same flower; (3) geitonogamy (GSP): 20 flowers were hand-pollinated with pollen from other flowers from the same individual; (4) crossing (CP): 20 flowers were hand-pollinated with pollen from another individual; (5) open-pollination (NP): 20 flowers were tagged and left for open pollination. Flowers subjected to hand-pollination were bagged with sulfuric acid paper bags and bagged again after hand-pollination. These experiments were conducted from 15 May to 30 May in 2013, 2014, and 2015. The fruit set of each treatment was counted two (July) and five months (October) later, and the seed set was counted 17 months later (October of the next year). For the natural fruit/seed set, we randomly selected 20 inflorescences to count and record the fruit/seed number per inflorescence. Analysis of variance (ANOVA) was performed to evaluate the difference of fruit/seed set for different treatments followed by Bonferroni post-hoc test. Fruit and seed set data were arcsine-transformed prior to analysis. The purpose of using the control and open pollination was to determine whether the pollen supplementation treatments affected fruit set [19]. We calculated an index of self-incompatibility (ISI) to determine the breeding system [20]. The index ranged from 0 to 1 and was the ratio between self and cross pollination, where 0.25 is considered the upper limit for self-incompatible species. Finally, the reproductive efficacy index (REI) was defined as the ratio between seed set from open-pollinated and cross-pollinated flowers; it also ranged from 0 to 1 and was used to estimate the relative efficacy of natural pollination [21]. The significance of ISI and REI was calculated to ensure that they were significantly different from zero based on 1000 bootstrap samples, using a 95% confidence interval, through bootstrap analysis using R version 3.4.1. (R Foundation for Statistical Computing, Vienna, Austria) Pollen tube growth experiments were also carried out to compare the compatibility of self- and cross-pollination. We bagged inflorescences on the day before the flowers opened and performed self- and cross-pollination treatments at 2, 4, 8, 12, 16, 24, 36, and 48 h. We picked 20 flowers per treatment, removed the styles and immediately fixed them in Formalin-acetic acid-alcohol (FAA) solution (formaldehyde, acetic acid, 70% ethanol at 5/5/90, v/v/v), transferred them to ethanol (70%) and stored them at 4 ◦C. Pollen tubes were measured according to the aniline blue method [18]. The growth rate (v) of the pollen tubes was calculated as the length of the pollen tube (Lpt), divided by the total style length (Ls) (v = Lpt/Ls).

2.4. Visitors To determine the pollinators and their visitation rates, counting of the visiting insect species was performed for all 20 randomly selected trees. The presence of floral visitors was recorded during sunny days from 15 May to 21 June in 2015 and 2016. The duration and frequency of visits and the behavior of the visitors were recorded. The selected trees were observed in two observation blocks, based on one-hour intervals, that is, between 08:00–09:00, 10:00–11:00, 13:00–14:00, and 16:00–17:00. The frequency of visitors was assessed in terms of flowers/minute. To estimate the difference between pollinators, counts of removed and deposited pollen grains were made subsequent to an initial visit by pollinators. Prior to visitation, 90 flowers from 20 trees were bagged with sulfuric acid paper bags to exclude visitors. The removed and deposited pollen grains Forests 2017, 8, 404 4 of 12 were counted after the flowers were first visited by the visitors according to the method described by Han et al. [22]. These samples were then transported to the laboratory to count the remaining pollen grains and those deposited on the stigmata. Sixty buds from ten trees were used to estimate the pollen production per flower. The anthers per flower were placed in a 5 mL centrifuge tube, and the quantity of pollen was determined according to the method described by Dafni (1992), based on 5 replicates per flower. Pollinator efficiency was calculated using the following formula:

MD pollinator efficiency = logMR (1) where MD is the mean number of pollen grains deposited on the stigmata, and MR is the mean number of removed pollen grains [23]. The capability of pollen removal and deposition was calculated using the following formula:

R the capability of pollen removal = the mean visiting frequency × the pollen production per flower (2) where R is the number of removed pollen grains; and

the capability of pollen deposition = the mean visiting frequency × D (3) where D is the number of pollen grains deposited on the stigmata. Analysis of variance (ANOVA) was performed to evaluate the difference in visitation frequency, pollinator efficiency, and the capability of pollen removal and deposition between pollinators followed by the Bonferroni post-hoc test.

3. Results

3.1. Floral Biology The flowering of Schima superba Garden. et Champ. occurs mainly from the middle of May to the middle of July. Flowers opened after sunrise at approximately 08:00 a.m., reached maximum aperture between 10:00–11:00, and withered in the afternoon on the fourth day (Figure1a,b). The flowers were arranged on a single racemose inflorescence containing an average of seven flowers (Table1). The average number of inflorescences per tree was 86.68. Flowers opened randomly in the inflorescence, and typically more than two flowers bloomed at the same time. The flowers are conspicuous, with a white, dialypetalous and actinomorphic corolla (mean length = 31.57 ± 2.69 mm), and have five connate white petals (mean length = 16.79 ± 2.18 mm). An average of approximately 80.2 stamens (mean length = 7.91 ± 2.42 mm, ranged from 6.9 to 12.2) are inserted around the style head, and the stigma is, on average, 8.30 ± 0.85 mm long (Figure1a, Table1). The flower is herkogamous with a mean value (mean length of stamens/mean length of stigma) of 0.95, and 96.7% of the examined flowers had longer stigmata than stamens. There also was a spatial separation between the stigmata and stamens in all flowers. The majority of the flowers presented viscous nectar with an average volume of 252.27 ± 16.67 µL. We observed nectar at the bottom of the filament, which smelled sweet after the flowers bloomed. The number of pollen grains and ovules produced per flower was 100,300 ± 47,056 and 15.00, respectively. Hence, the P/O ratio was 6686.67.

Table 1. Floral characteristics of Schima superba Garden. et Champ (S.E.).

Items NIP NFI LC (mm) LP (mm) LS (mm) LOS (mm) Pollen Grains Ovules Pollen-Ovule Ratio Mean (SE) 86.68 (21.42) 7 (2) 31.57 (2.69) 16.79 (2.18) 8.30 (0.85) 7.91 (2.42) 100,300 (47,056) 15.00 6686.67 Range 40.50–147.55 4–11 27.86–35.12 11.73–20.35 6.80–9.60 4.01–12.25 92,104–165,105 - - N 30 30 60 60 60 60 30 30 30 Note: N, sample size; NIP, number of inflorescence/plant; NFI, number of flowers/inflorescence; LC, length of corolla; LP, length of petal; LS, length of stigma; LOS, length of stamen. Forests 2017, 8, 404 4 of 12

described by Han et al. [22]. These samples were then transported to the laboratory to count the remaining pollen grains and those deposited on the stigmata. Sixty buds from ten trees were used to estimate the pollen production per flower. The anthers per flower were placed in a 5 mL centrifuge tube, and the quantity of pollen was determined according to the method described by Dafni (1992), based on 5 replicates per flower. Pollinator efficiency was calculated using the following formula:

pollinator efficiency = (1) where MD is the mean number of pollen grains deposited on the stigmata, and MR is the mean number of removed pollen grains [23]. The capability of pollen removal and deposition was calculated using the following formula: the capability of pollen removal = the mean visiting frequency × the pollen production per flower (2) where R is the number of removed pollen grains; and

the capability of pollen deposition = the mean visiting frequency × (3) where D is the number of pollen grains deposited on the stigmata. Analysis of variance (ANOVA) was performed to evaluate the difference in visitation frequency, pollinator efficiency, and the capability of pollen removal and deposition between pollinators followed by the Bonferroni post-hoc test.

3. Results

3.1. Floral Biology The flowering of Schima superba Garden. et Champ. occurs mainly from the middle of May to the middle of July. Flowers opened after sunrise at approximately 08:00 a.m., reached maximum aperture between 10:00–11:00, and withered in the afternoon on the fourth day (Figure 1a,b). The flowers were arranged on a single racemose inflorescence containing an average of seven flowers (Table 1). The average number of inflorescences per tree was 86.68. Flowers opened randomly in the inflorescence, and typically more than two flowers bloomed at the same time. The flowers are conspicuous, with a white, dialypetalous and actinomorphic corolla (mean length = 31.57 ± 2.69 mm), and have five connate white petals (mean length = 16.79 ± 2.18 mm). An average of approximately 80.2 stamens (mean length = 7.91 ± 2.42 mm, ranged from 6.9 to 12.2) are inserted around the style head, and the stigma is, on average, 8.30 ± 0.85 mm long (Figure 1a, Table 1). The flower is herkogamous with a mean value (mean length of stamens/mean length of stigma) of 0.95, and 96.7% of the examined flowers had longer stigmata than stamens. There also was a spatial separation between the stigmata and stamens in all flowers. The majority of the flowers presented viscous nectar with an average volume of 252.27 ± 16.67 μL. We observed nectar at the bottom of the filament, which Forestssmelled2017, 8, 404 sweet after the flowers bloomed. The number of pollen grains and ovules produced per5 of 12 flower was 100,300 ± 47056 and 15.00, respectively. Hence, the P/O ratio was 6686.67.

Forests 2017, 8, 404 5 of 12

Table 1. Floral characteristics of Schima superba Garden. et Champ (S.E.).

Pollen- LOS Items NIP NFI LC (mm) LP (mm) LS (mm) Pollen grains Ovules Ovule (mm) Ratio Mean (SE) 86.68 (21.42) 7 (2) 31.57 (2.69) 16.79 (2.18) 8.30 (0.85) 7.91 (2.42) 100300 (47056) 15.00 6686.67 Range 40.50–147.55Figure 1. 4–11The flowers27.86–35.12 and pollinators11.73–20.35 of6.80–9.60 Schima 4.01–12.25 superba Garden.92104–165105 et Champ. - - N Figure 30 1. The30 flowers60 and pollinators 60 of60Schima superba60 Garden. 30 et Champ.30 30 Note: N, sample size; NIP, number of inflorescence/plant; NFI, number of flowers/inflorescence; LC, 3.2. Pollen Viabilitylength of andcorolla; Stigma LP, length Receptivity of petal; LS, length of stigma; LOS, length of stamen. The3.2. viability Pollen Viability of pollen and Stigma showed Receptivity a sharp decreasing trend: the viability was relatively stable initially; thereafter, theThe viability viability ofof thepollen pollen showed decreased a sharp decreasing rapidly to trend: 5.45% the at viability 09:00 onwas Day relatively 4 (Figure stable2 ). Based on initially; thereafter, the viability of the pollen decreased rapidly to 5.45% at 09:00 on Day 4 (Figure 2). the daily average viability, the percentage of viable pollen (in decreasing order) was observed on Based on the daily average viability, the percentage of viable pollen (in decreasing order) was Day 1 >observed Day 2 > on Day Day 3 1 >> Day 2 4> .Day The 3 > stigma Day 4. The is papillose,stigma is papillose, wet after wet pollinationafter pollination and and situated situated at a similar height asat the a similar anthers. height The as stigma the anthers. receptivity The stigma started receptivity before started anther before dehiscence, anther dehiscence, which points which to a slightly retainedpoints protogynous to a slightly type retained of dichogamyprotogynous type in S. of dichogamy superba. The in S. stigmasuperba. The remained stigma remained highly highly receptive for the receptive for the entirety of the flowers’ lifetime (Figure 2). entirety of the flowers’ lifetime (Figure2).

100 Receptivity 100 Viability

80 80

60 60

Viability (%) 40 40 Receptivity (%)

20 20

0 0 -- 1d 0900 1d 1500 1d 2100 2d 0900 2d 1500 2d 2100 3d 0900 3d 1500 3d 2100 4d 0900 4d 1500 4d 2100 Time Figure 2. Pollen viability and stigma receptivity in Schima superba (S.E. percentage per day). Figure 2. Pollen viability and stigma receptivity in Schima superba (S.E. percentage per day). 3.3. Breeding System 3.3. Breeding System No fruits were obtained from the bagged flowers, indicating the absence of agamospermy for this Nospecies. fruits wereThe fruit obtained and seed fromset of cross-pollination the bagged flowers, (CP) were indicating significantly the higher absence than geitonogamy of agamospermy self- for this pollination (GSP) (fruit set: F1, 43 = 273.724, P < 0.001, seed set: F1, 27 = 407.019, P < 0.001 for seed set) and species. The fruit and seed set of cross-pollination (CP) were significantly higher than geitonogamy self-pollination with the same flower (SFSP) (fruit set: F1, 43 = 265.224, P < 0.001, seed set: F1, 27 = 407.011, self-pollinationP < 0.001) (GSP) (Figure (fruit3). The set: reproductiveF1, 43 = 273.724,efficacy indexP < (REI) 0.001, of seedS. superba set: wasF1, 27low= (0.56 407.019, ± 0.03,P which< 0.001 for seed set) andranged self-pollination from 0.50 to 0.63 with at the the 95% same confidence flower interv (SFSP)al), and (fruitthe fruit set: and seedF1, 43 set= from 265.224, open-pollinationP < 0.001, seed set: (OP) was significantly lower than in the CP treatment (fruit set: F1, 43 = 23.145, P < 0.001, seed set: F1, 27 = F1, 27 = 407.011, P < 0.001) (Figure3) . The reproductive efficacy index (REI) of S. superba was low ± 40.633, P < 0.001). The CP treatment was more successful than the SP and OP treatments: maximum (0.56 0.03fruit, (41.99%) which rangedand seed from (11.66%) 0.50 setting to 0.63 was at observed the 95% in confidence the CP treatment, interval), followed and theby the fruit NP and seed set from open-pollinationtreatment (27.70% (OP)and 6.57%, was respectively). significantly The lower GSP treatment than in yielded the CP the treatment least fruit (1.91%) (fruit and set: seedF1, 43 = 23.145, P < 0.001(0.20%),, seed which set: F were1, 27 =equivalent 40.633, toP the< 0.001). results obtained The CP with treatment the SFSP wastreatment more (fruit successful set, 2.05%; thanseed the SP and OP treatments:set, 0.40%), maximum and there was fruit no (41.99%)significant anddifference seed between (11.66%) GSP setting and SFSP was (fruit observed set, F1, 43 = in0.084, the P CP = treatment, 0.773; seed set, F1, 27 = 0.042, P = 0.184). The fruit abortion was very high; the fruit abortion rates of the followedSFSP, by theGSP, NP CP and treatment NP treatments (27.70% were and 88.8%, 6.57%, 88.9%, respectively).32.3%, and 33.1%, The respectively GSP treatment (Figure 3). yielded the least fruit (1.91%) and seed (0.20%), which were equivalent to the results obtained with the SFSP treatment (fruit set, 2.05%; seed set, 0.40%), and there was no significant difference between GSP and SFSP Forests 2017, 8, 404 6 of 12

(fruit set, F1, 43 = 0.084, P = 0.773; seed set, F1, 27 = 0.042, P = 0.184). The fruit abortion was very high; the fruit abortion rates of the SFSP, GSP, CP and NP treatments were 88.8%, 88.9%, 32.3%, and 33.1%, respectivelyForests 2017 (Figure, 8, 404 3). 6 of 12

80 80 Forests 2017, 8, 404 6 of 12 70 70

60 60

50 80 8050

40 70 7040 30 30 60 60 Fruit set, July set, Fruit

Fruit set, October set, Fruit 5020 20 50 4010 10 40 30 30 0

Fruit set, July set, Fruit 0

NP CP GSP SFSP October set, Fruit 20 NP CP GSP SFSP 20 80 10 10 70 0 0 60 NP CP GSP SFSP NP CP GSP SFSP 80 50 70 40 60

Seed set 30 50 20 40

10Seed set 30 0 20 NP CP GSP SFSP 10 0 Figure 3. The effect of pollinationNP CP treatments GSP SFSP on the fruit and seed set of Schima superba (S.E.). Note: Figure 3. The effect of pollination treatments on the fruit and seed set of Schima superba (S.E.). Note: NP, open-pollinations;NP, open-pollinations; CP, cross-pollination;CP, cross-pollination; GSP, geitonogamousGSP, geitonogamous self-pollination; self-pollination; SFSP, SFSP, self-pollination self- Figure 3. The effect of pollination treatments on the fruit and seed set of Schima superba (S.E.). Note: withpollination the same flower. with the same flower. NP, open-pollinations; CP, cross-pollination; GSP, geitonogamous self-pollination; SFSP, self- The pollenThepollination pollen tube tube with growth growth the same experiments experiments flower. showed showed that pollen pollen growth growth after after the the crossing crossing treatment treatment was was more rapid than in the self-pollination treatment, as assessed during the first 2 h (9.82% vs. 2.48%). more rapidThe than pollen in the tube self-pollination growth experiments treatment, showed that as pollen assessed growth during after the the crossing first 2 htreatment (9.82% was vs. 2.48%). TheseThese twomore two treatments rapid treatments than in reached the reached self-pollination their their greatest greatest treatment, differencediffe asrence assessed at at 1212 during h h (81.23% (81.23% the first vs. vs.2 49.45%), h (9.82% 49.45%), andvs. 2.48%). andthen thenthe the bottombottomThese of the of two stylesthe treatmentsstyles was was reached reachedreached after their after 36 greatest36 h h (Figures(Figures difference 44 andand at 55). ).12 Ath (81.23%48 48 h, h, the the vs. pollen pollen 49.45%), tube tube and of the ofthen thecrossing the crossing treatments reached the ovary, but the pollen tube of the self-pollinated flowers did not (Figure 4). treatmentsbottom reached of the styles the ovary,was reached but theafter pollen 36 h (Figures tube of4 and the 5). self-pollinated At 48 h, the pollen flowers tube of didthe crossing not (Figure 4). The self-incompatibility index (ISI) was estimated to be 0.95 ± 0.01 (0.93–0.97 at 95% confidence The self-incompatibilitytreatments reached the index ovary, (ISI) but wasthe pollen estimated tube of to the be self-pollinated 0.95 ± 0.01 flowers (0.93–0.97 did not at (Figure 95% confidence 4). interval),The self-incompatibility suggesting that S. index superba (ISI) behaved was estimated mostly asto abe late-acting 0.95 ± 0.01 self-incompatible (0.93–0.97 at 95% (LSI) confidence species. interval),interval), suggesting suggesting that thatS. superba S. superbabehaved behaved mostly mostly as as a a late-acting late-acting self-incompatible self-incompatible (LSI) species. (LSI) species.

FigureFigure 4. 4.Pollen Pollen tube tube growth growth of of Schima Schima superbasuperba for self-self- andand cross-pollination cross-pollination treatments treatments in instyle style and and Figure 4. Pollen tube growth of Schima superba for self- and cross-pollination treatments in style and ovary. ovary.ovary. Note: Note: (A ()A pollen) pollen tubes tubes resulting resulting fromfrom cross-pollination grew grew to to the the medial medial style style at at12 12h; (h;B )( B) Note: (A) pollen tubes resulting from cross-pollination grew to the medial style at 12 h; (B) pollen tubes pollenpollen tubes tubes resulting resulting from from self-pollination self-pollination grew to thethe medialmedial style style at at 12 12 h; h; (C ()C pollen) pollen tubes tubes from from resultingcross-pollinationcross-pollination from self-pollination grew grew to to the grewthe base base to of theof the the medial stylestyle at style 36 h; at ((DD 12)) pollen h;pollen (C) tubes pollentubes resulting resulting tubes from from from cross-pollinationself-pollination self-pollination grew to thegrew basegrew to of tothe the the base style base of atof the the 36 style h;style (D at at) 36 pollen 36 h h with with tubes partpart resulting retardation from (shown(shown self-pollination by by red red arrows); arrows); grew (E ()E cross-pollinated to) cross-pollinated the base of the style at 36pollen h withpollen tubes part tubes retardationentered entered the the ovary; (shown ovary; ( F(F by)) nonenone red of arrows);of thethe self-pollinated (E) cross-pollinated pollen pollen tubes tubes pollen entered entered tubes the the ovary. entered ovary. SEM theSEM ovary; resolution of 200 μm. (F) noneresolution of the self-pollinatedof 200 μm. pollen tubes entered the ovary. SEM resolution of 200 µm.

Forests 2017,, 88,, 404404 7 of 12

Self-pollination 100 Cross-pollination

80

60

40 The growth rate/% growth The

20

0 2 4 8 12 16 24 36 48 Hours after hand-pollination (h)

Figure 5. The pattern of pollen tube growth in the styles of Schima superba under two pollinationpollination treatments (S.E.).(S.E.)

3.4. Pollinator Visitation A large number of ApisApis ceranacerana ceranacerana Fabricius,Fabricius, Protaetia brevitarsis Lewis and Popillia mutans Newman were observed within the whole floweringflowering phase of S.S. superbasuperba.. Therefore,Therefore, wewe determineddetermined that the three insects were the main pollinators of S. superba throughthrough observation. observation. In In the the seed seed orchard, orchard, Pieris rapaerapae, Nephargynnis, Nephargynnis anadyomene anadyomene, Epicopeia, Epicopeia mencia menciaand Musca and Musca domestica domesticawere occasionally were occasionally observed withobserved flower-visiting with flower-visiting behavior; behavior; however, however, they were they not were the mainnot the pollinators main pollinators because because of very of small very quantitysmall quantity in the in seed the orchard.seed orchard.A. cerana A. ceranalanded landed on any on partany part of flowers of flowers and and drew drew the nectarthe nectar from from the bottomthe bottom of the of the stamen stamen using using their their mouthparts mouthparts (Figure (Figure1c,d). 1c,d).P. P. brevitarsis brevitarsisand andP. P. mutansmutans hadhad similar visiting behaviors. The beetles were rewarded wi withth pollen found in the staminode and and inner petals. petals. The pollenpollen adheredadhered to to the the mouthparts, mouthparts, feet, feet, and and sternum sternum of the of beetlesthe beetles as they as fed,they though fed, though some pollensome alsopollen adhered also adhered to the backto the of back the animalof the animal (Figure (Fig1e,f).ure We 1e,f). also We observed also observed that the that beetles the beetles would would often tryoften to try dive to theirdive mouthpartstheir mouthparts into the into nectary the nectary (at the (at bottom the bottom of the of stamens) the stamens) to suck to suck nectar. nectar. Their Their legs, chest,legs, chest, and abdomen and abdomen could could touch touch the stigma the stigma when theywhen walked they walked on the flowers;on the flowers; thus, we thus, believe we thatbelieve the beetles’that the pollinationbeetles’ pollination strategy isstrategy abdominal is abdomina pollination.l pollination. There was There a major was difference a major indifference the abundance, in the identity,abundance, and identity, diversity ofand pollinators diversity between of pollinators plants. Thebetween average plants. pollinator The visitaverage (abundance, pollinator identity, visit and(abundance, diversity) identity, per plant and was divers 1 (0–4)ity) for perA. plant cerana was, 1.1 1 (0–8) (0–4) for forP. A. brevitarsis cerana, 1.1, and (0–8) 10.2 for (5–18) P. brevitarsis for P. mutans, and. 10.2 (5–18)The floral for measurementP. mutans. results shed some light on the flower-pollinator interactions (Figures6 and7) . The largeThe floral corolla measurement made it possible results for the shed putative some pollinatorslight on the to flower-pollinator land on and enter interactions the flower. The (Figures highest 6 visitingand 7). The frequency large corolla occurred made between it possible 10:00–11:00 for the pu (Figuretative6 ).pollinators There were to land no significant on and enter differences the flower. in The highest visiting frequency occurred between 10:00–11:00 (Figure 6). There were no significant visiting frequency between P. brevitarsis and P. mutans (F1, 129 = 9.283, P = 0.053). Nevertheless, the visiting differences in visiting frequency between P. brevitarsis and P. mutans (F1, 129 = 9.283, P = 0.053). frequency of A. cerana was significantly higher than P. brevitarsis (F1, 127 = 53.282, P < 0.001) and P. mutans Nevertheless, the visiting frequency of A. cerana was significantly higher than P. brevitarsis (F1, 127 = (F1, 127 = 43.191, P < 0.001) (Figure7). This means that A. cerana had absolute superiority in visiting frequency53.282, P < compared 0.001) and with P. mutans the two ( beetles,F1, 127 = as43.191, evidenced P < 0.001) by their (Figure ability 7). to This visit means more flowers that A. than cerana beetles had inabsolute a given superiority amount of in time. visiting The pollinatorfrequency efficiencycompared of withP. mutans the twowas beetles, slightly as evidenced higher than by the their two ability other pollinatorsto visit more (Figure flowers7). than There beetles was no in significanta given amount difference of time. between The pollinatorP. brevitarsis efficiencyand P. of mutans P. mutanswith was slightly higher than the two other pollinators (Figure 7). There was no significant difference respect to the capability of pollen removal (F1, 64 = 67.403, P = 0.672) and deposition (F1, 64 = 31.632, Pbetween= 1.000) P.. Nevertheless, brevitarsis and the P. capabilitymutans with of pollenrespect removal to the capability and deposition of pollen of A. removal cerana was (F1, 64 significantly = 67.403, P = 0.672) and deposition (F1, 64 = 31.632, P = 1.000). Nevertheless, the capability of pollen removal and higher than P. brevitarsis (capability of pollen removal: F1, 54 = 116.122, P < 0.001, capability of pollen deposition of A. cerana was significantly higher than P. brevitarsis (capability of pollen removal: F1, 54 deposition: F1, 54 = 18.063, P < 0.001) and P. mutans (capability of pollen removal: F1, 59 = 78.512, P < 0.001, = 116.122, P < 0.001, capability of pollen deposition: F1, 54 = 18.063, P < 0.001) and P. mutans (capability capability of pollen deposition: F1, 59 = 10.693, P < 0.001) (Figure7). of pollen removal: F1, 59 = 78.512, P < 0.001, capability of pollen deposition: F1, 59 = 10.693, P < 0.001) (Figure 7).

ForestsForests 2017 2017, ,8 8, ,404 404 88 of of 12 12 Forests 2017, 8, 404 8 of 12

1010 Apis Apis cerana cerana crenan crenan Fabricius Fabricius Protaetia Protaetia brevitarsis brevitarsis Lewis Lewis Popillia Popillia mutans mutans Newman Newman 88

66

44 Visiting frequency Visiting frequency 22

00

-2-2 08:00-09:0008:00-09:00 10:00-11:00 10:00-11:00 13:00-14:00 13:00-14:00 16:00-17:00 16:00-17:00

TimeTime of of day day

FigureFigure 6. 6. Pollinator Pollinator visitation visitation frequency frequency for for Schima Schima superbasuperba (S.E.). (S.E.).

88 Pollinator efficiency Pollinator efficiency 0.600.60 Visiting Visiting frequency frequency per per minute minute 66 0.580.58 44 0.560.56

2 2 0.540.54 Pollinator efficiency Pollinator efficiency 00 0.520.52 Visiting frequency per minute Visiting frequency per minute

600600 The The capability capability of of pollen pollen removal removal 25002500 500500 The The capability capability of of pollen pollen deposition deposition 20002000 400400 15001500 300300 10001000 200200 500500 100100

00 00 The capability of pollen removal The capability of pollen removal The capability of pollen deposition The capability of pollen deposition A.A. cerana cerana cerana cerana Fabridus Fabridus P.P. brevitarsis brevitarsis Lewis Lewis P.P. mutans mutans Newman Newman

FigureFigure 7.7. TheThe general generalgeneral visiting visitingvisiting frequency, frequency,frequency, pollinator pollinator efficiency, efficiency,efficiency, capability capability of of pollenpollen removalremoval andand depositiondeposition forfor three three pollinators pollinators after after the the firstfirst first visitationvisitation visitation ofof of aa a SchimaSchima Schima superbasuperba superba flowerflower flower (S.E.).(S.E.) (S.E.)

4.4. Discussion Discussion PhenologicalPhenological and andand reproductive reproductivereproductive biology biologybiology studies studiesstudies are arare usefule usefuluseful for forfor planning planningplanning conservation conservationconservation strategies strategiesstrategies as wellasas wellwell as formulating asas formulatingformulating measures measuresmeasures for forcultivatingfor cultivatingcultivating such suchsuch species speciesspecies at a at largeat aa largelarge scale scalescale [24]. [24].[24]. The The moreThe moremore flowers flowersflowers and theandand larger thethe largerlarger flower flowerflower diameter diameterdiameter the plants thethe plants have,plants the have,have, more ththe pollinatorse moremore pollinatorspollinators are attracted, areare attracted,attracted, which leads whichwhich to superiorleadsleads toto fruitsuperiorsuperior set and fruit fruit pollen set set and and output pollen pollen rate output output [25–27 ra ra].tete The [25–27]. [25–27]. flower The The diameter fl flowerower ofdiameterdiameterS. superba of of S. Gardn.S. superba superba et Gardn. Champ.Gardn. et et exceeded Champ. Champ. 27.86exceededexceeded mm . 27.8627.86 This mm. speciesmm. ThisThis belongs speciesspecies to belongsbelongs species toto with speciespecie largess withwith flowers largelarge according flowersflowers accordingaccording to the technical toto thethe standardtechnicaltechnical ofstandardstandard Abe [28 of],of and AbeAbe this [28],[28], may andand attract thisthis may pollinatorsmay attractattract to pollinatorspollinators visit flowers toto andvisitvisit increase flowersflowers pollination andand increaseincrease opportunities. pollinationpollination opportunities.opportunities. The The natural natural flower flower life life ranged ranged from from 4 4 to to 5 5 days, days, which which categorizes categorizes the the plant plant as as a a longer longer

Forests 2017, 8, 404 9 of 12

The natural flower life ranged from 4 to 5 days, which categorizes the plant as a longer flowering type [28]; this enhances the opportunity of floral visits and ensures the stability of pollen flow. Flowers of S. superba have abundant and well-exposed rewards that are potentially easily exploited by a wide array of flower visitors: nectar can be reached by both short and long mouthparts. Several traits contribute to attracting beetles and protecting the ovary from being eaten by them: flowers that are white, larger and gathered into an inflorescence with an inferior ovary [29]. These traits are potentially a result of mutualism between beetles and plants [30]. Protogyny in S. superba may be another adaptation to pollination by beetles [29]. Anthesis in S. superba is diurnal, beginning at approximately 08:00–09:00 am after sunlight irradiation. In sexual reproduction, the floral syndrome and pollination behavior are reciprocal adaptations, whereas a high amount of viable pollen and many receptive stigmata are prerequisites for effective pollination. The duration of pollen viability and receptivity directly affects the effectiveness of pollination and the success rate of fertilization at different flowering stages [31]. In the current study, pollen viability decreased from the first day of flower opening to the time at which the pollen fell off the flower, which may be associated with the loss of water upon sunlight exposure [16]. Therefore, we predicted that rapid inactivation of pollen was one of the primary reasons for the low fruit set of S. superba. In contrast, higher receptivity within a flower’s natural life (that is, with high female reproductive fitness) could enhance the fruit set of S. superba under adverse environmental conditions as well as improve the number of pollinators and pollination efficiency. There was a major difference in the abundance, identity, and diversity of pollinators between plants, which could be the reason for lower fruit set in some plants. Therefore, we can improve the seed production of S. superba seed orchards through artificial cultivation of bees. According to the high calculated index (0.95) of self-incompatibility (ISI) and the high observed pollen/ovule ratio (P/O) (6686.67), we believe that S. superba is mostly self-incompatible with obligatory outbreeding [21,32]. This, together with the results of the pollen tube growth experiments, suggests that incompatibility may be late-acting self-incompatibility (LSI) [33], and the incompatibility may occur in the ovary. In the present study, pollen tubes reached the base of the style 36 h after self-pollination and cross-pollination, and the growth rate of the crossed tubes was slightly higher than that of the self-tubes, similar to what has been described for other plants of the same family, such as Camellia oleifera Abel. and Camellia sinensis (L.) O. Ktze. [34]. Furthermore, the pollen tubes did not penetrate the ovule after self-pollination (Figure4E). This may be the result of the generation of resistant materials in the ovule or micropyle (e.g., callose) that prevented the self-pollinated pollen tubes from penetrating the ovule [34]. Fruit and seed set is the standard by which mating success is estimated. The extremely low seed set of geitonogamous and autogamous self-pollination (GSP and SFSP) indicated that the breeding system of S. superba is obligate xenogamy, similar to the results from the ISI and P/O. This breeding system can prevent a decline in seed genetic quality due to self-pollination in a seed orchard. The herkogamy of S. superba could also reduce self-pollination and prevent inbreeding depression [35–37]. The fruit and seed set resulting from cross-pollination (CP) is higher than that resulting from open-pollination (OP), suggesting that the seed orchard was probably pollinator-limited [38]. It also showed that artificial pollination can significantly improve the fruit and seed set of S. superba. The pollen limitation of S. superba may be due to both the quantity and quality of the pollen deposited on the stigmata, as we detected a rapid devitalization of pollen (Figure2). The low reproductive efficacy index (0.66) of S. superba may be related to both its late-acting self-incompatibility system and to the beetles’ infrequent visits, which, in turn, may have resulted in pollen limitation [39]. This explanation is also confirmed by the observed significantly higher fruiting success of cross-pollination compared with open-pollination. We also detected a higher decreasing rate in the cross- and open-pollination treatments, suggesting that resource limitation may be an important factor because of the high amount of humidity and nutrients demanded during a growth and development period as long as 1.5 years. In the future, to reduce the percentage of fruit abortion, improved management and adequate fertilization methods should be implemented to ensure the necessary humidity and nutrients required for adequate fruit growth. Forests 2017, 8, 404 10 of 12

Mapping floral characters and pollinators onto phylogenies shows that the relationship between flowers and their pollinators is prone to coadaptation [40]. The flowers of S. superba show many characteristics of insect pollination, such as a strong smell, an abundance of nectar and a stigma well separated from the nectary. We found that S. superba is pollinated by three insects (Apis cerana cerana Fabricius, Protaetia brevitarsis Lewis and Popillia mutans Newman), unlike other Theaceae plants such as Schima wallichii (DC.) Korth [6], Ternstroemia laevigata and Ternstroemia dentate [13]. Based on the pollen viability over time (Figure2), it is plausible that the three pollinators are the primary and key pollinators of S. superba [16]. P. mutans is more abundant than P. brevitarsis and A. cerana; however, the visitation frequency of the two beetles is significantly lower than that of A. cerana, which may limit fruit/seed production. Therefore, A. cerana is an important and efficient pollinator of S. superba. The pollen removal of A. cerana in this study was significantly lower than that reported for single visits by Apis to other plants [41]. This observation could be explained by the large flower size and numerous pollen grains of S. superba. Thomson found that larger Bombus queens made more contact with Erythronium grandiflorum Pursh (Liliaceae) stigmata [42], and Snow also reported that larger bees deposited more pollen on Cassia flowers than smaller bees [43]. Although removal and deposition may increase with body size, in the present study, the smaller P. mutans removed and deposited more pollen than P. brevitarsis (which has a larger body size than P. mutans), suggesting that small beetles could potentially remove and deposit more or as much pollen as larger species. Suzuki hypothesized that flowers had a higher pollinator visiting frequency, and the flowers could increase the amount of fruit set [44]. The amount of removed and deposited pollen grains by P. mutans is numerically higher than P. brevitarsis and A. cerana but not significantly so. Moreover, the capability of pollen removal of A. cerana was significantly higher than the two beetles, i.e., 21.58 and 6.83 times higher than P. brevitarsis and P. mutans, respectively. The capability of pollen deposition of A. cerana was also significantly higher than the two beetles, i.e., 17.88 and 4.67 times higher than P. brevitarsis and P. mutans, respectively. In general, a higher visitation frequency translates to a higher fruit set [41]. Thus, we believe A. cerana was the most efficient pollinator of S. superba.

5. Conclusions In summary, we investigated the floral biology, breeding system and pollination ecology of Schima superba Gardn. et Champ. The breeding system of S. superba is characterized as late-acting self-incompatible. The viability of pollen decreased rapidly, but the stigmata maintained high receptivity during the flowers’ life spans. Three visitors were defined as pollinators: A. cerana cerana Fabricius appeared to be the most effective pollinator. Finally, we confirm that the seed production of S. superba is frequently limited by pollinators, fruit abortion, and pollen quality. A comprehensive analysis revealed that seed production could be improved through the artificial cultivation of bees in an S. superba seed orchard.

Acknowledgments: Research was supported by the National Science & Technology Pillar Program during the 12th Five-year Period (2012BAD01B04), and Zhejiang Science and Technology Major Program on Agricultural New Variety Breeding, China (2016C02056-3). Author Contributions: Hanbo Yang and Rui Zhang conceived and designed the experiments; Hanbo Yang performed the experiments; Hanbo Yang and Ping Song analyzed the data; Zhichun Zhou contributed reagents/materials/analysis tools; and Hanbo Yang drafted the manuscript. Conflicts of Interest: The authors declare that there are no financial or personal relationships with other people or organizations that could inappropriately influence this work, and that there are no professional or other personal interests of any nature or in any product, service and/or company, which could influence the positions presented herein or affect the review of the manuscript.

References

1. Charlesworth, D. Evolution of plant breeding systems. Curr. Biol. 2006, 16, 726–735. [CrossRef][PubMed] 2. Montesinos, D.; Oliveira, P. Reproductive ecology of buzz-pollinated Ouratea spectabilis trees (Ochnaceae) in Brazilian cerrados. Web Ecol. 2015, 14, 79–84. [CrossRef] Forests 2017, 8, 404 11 of 12

3. Gong, X.; Guan, B.C.; Zhou, S.L.; Ge, G. Reproductive biology of the rare plant, Dysosma pleiantha (Berberidaceae): Breeding system, pollination and implications for conservation. Pak. J. Bot. 2015, 47, 951–957. 4. Iwata, T.; Nagasaki, O.; Ishii, H.S.; Ushimaru, A. Inflorescence architecture affects pollinator behaviour and mating success in Spiranthes sinensis (Orchidaceae). New Phytol. 2012, 193, 196–203. [CrossRef][PubMed] 5. Aronne, G.; Buonanno, M.; Micco, V.D. Reproducing under a warming climate: Long winter flowering and extended flower longevity in the only Mediterranean and maritime primula. Plant Biol. 2015, 17, 535–544. [CrossRef][PubMed] 6. Khanduri, V.P.; Sharma, C.M.; Kumar, K.S.; Ghildiyal, S.K. Annual variation in flowering phenology, pollination, mating system, and pollen yield in two natural populations of Schima wallichii (DC.) Korth. Sci. World J. 2013, 2013, 1653–1656. [CrossRef][PubMed] 7. Delmas, C.E.L.; Escaravage, N.; Cheptou, P.-O.; Charrier, O.; Ruzafa, S.; Winterton, P.; Pornon, A. Relative impact of mate versus pollinator availability on pollen limitation and outcrossing rates in a mass-flowering species. Plant Biol. 2014, 17, 209–218. [CrossRef][PubMed] 8. Li, J.; Pan, B.; Ge, R.; Fang, H. Pollination insects and their flower-visiting behaviors on endangered plant Amydalus pedunculata. Chin. J. Ecol. 2011, 30, 1370–1374. (In Chinese) 9. Sigrist, M.R.; Sazima, M. Phenology, reproductive biology and diversity of buzzing bees of sympatric dichorisandra species (Commelinaceae): Breeding system and performance of pollinators. Plant Syst. Evol. 2014, 301, 1–11. [CrossRef] 10. Barriault, I.; Gibernau, M.; Barabe, D. Flowering period, thermogenesis, and pattern of visiting insects in Arisaema triphyllum (Araceae) in Quebec. Botany 2009, 87, 324–329. [CrossRef] 11. Young, H.J. Diurnal and nocturnal pollination of Silene alba (Caryophyllaceae). Am. J. Bot. 2002, 89, 433–440. [CrossRef][PubMed] 12. Stevens, P.F.; Dressler, S.; Weitzman, A.L. Theaceae. In Flowering Plants Dicotyledons; Springer: Berlin, Germany, 2004; pp. 463–471. 13. Bittrich, V.; Amaral, M.C.E.; Melo, G.A.R. Pollination biology of Ternstroemia laevigata and T. dentata (Theaceae). Plant Syst. Evol. 1993, 185, 1–6. [CrossRef] 14. Caddell, G.M. Pollination, Mating System, and Distribution of Genetic Variation within Populations of Camellia japonica L. (Theaceae). Ph.D. Thesis, The University of North Carolina, Chapel Hill, NC, USA, 1989. 15. Zhang, R.; Zhou, Z.C.; Luo, W.J.; Wang, Y.; Feng, Z.P. Effects of nitrogen deposition on growth and phosphate efficiency of Schima superba of different provenances grown in phosphorus-barren soil. Plant Soil 2013, 370, 435–445. [CrossRef] 16. Ekeke, C.; Agbagwa, I.O. Breeding and pollination biology of Combretum constrictum (benth) laws. (Combretaceae). Trop. Plant Biol. 2015, 8, 51–59. [CrossRef] 17. Chinese Academy of Sciences Editorial Board of . Flora of China; Science Press: Beijing, China, 1998; Volume 49, p. 224. 18. Dafni, A. Pollination Ecology: A Practical Approach; Oxford University Press: Oxford, UK, 1992; Volume 46, pp. 250–478. 19. Zimmerman, M.; Pyke, G.H. Reproduction in polemonium: Assessing the factors limiting seed set. Am. Nat. 1988, 131, 723–738. [CrossRef] 20. Bullock, S.T. Breeding systems in the flora of a tropical deciduous forest in Mexico. Biotropica 1985, 17, 287–301. [CrossRef] 21. Zapata, T.R. Plant reproductive ecology of a secondary deciduous tropical forest in Venezuela. Biotropica 1978, 10, 221–230. [CrossRef] 22. Han, Y.; Dai, C.; Yang, C.-F.; Wang, Q.-F.; Motley, T.J. Anther appendages of Incarvillea trigger a pollen- dispensing mechanism. Ann. Bot. 2008, 102, 473–479. [CrossRef][PubMed] 23. Harder, L.D.; Wilson, W.G. Floral evolution and male reproductive success: Optimal dispensing schedule for pollen dispersal by animal-pollinated plants. Evol. Ecol. 1994, 8, 542–559. [CrossRef] 24. Bernardello, G.; Crawford, D.J. A survey of floral traits, breeding systems, floral visitors, and pollination systems of the angiosperms of the Juan Fernández islands (Chile). Bot. Rev. 2001, 67, 255–308. [CrossRef] 25. Podolsky, R.D. Strange floral attractors: Pollinator attraction and the evolution of plant sexual systems. Science 1992, 258, 791–793. [CrossRef][PubMed] 26. Ishii, H.S.; Sakai, S. Temporal variation in floral display size and individual floral sex allocation in racemes of Narthecium asiaticum (Liliaceae). Am. J. Bot. 2002, 89, 441. [CrossRef][PubMed] Forests 2017, 8, 404 12 of 12

27. Brunet, J.; Sweet, H.R. Impact of insect pollinator group and floral display size on outcrossing rate. Evolution 2006, 60, 234–246. [CrossRef][PubMed] 28. Abe, T. Flowering phenology, display size, and fruit set in an understory dioecious shrub, Aucuba japonica (Cornaceae). Am. J. Bot. 2001, 88, 455–461. [CrossRef][PubMed] 29. Bawa, K.S.; Beach, J.H. Evolution of sexual systems in flowering plants. Ann. Missouri Bot. Gard. 1981, 68, S243. [CrossRef] 30. Gottsberger, U.D.G. Some aspects of beetle pollination in the evolution of flowering plants. In Flowering Plants; Springer: Vienna, Austria, 1977; pp. 211–226. 31. Amyc, D.; Rosanna, F. Floral development, stigma receptivity and pollen viability in eight nolana (Solanaceae) species. Euphytica 2010, 174, 105–117. 32. Cruden, R.W. Pollen-ovule ratios: A conservative indicator of breeding systems in flowering plants. Evolution 1977, 31, 32–46. [CrossRef][PubMed] 33. Gibbs, P.E. Late-acting self-incompatibility—The pariah breeding system in flowering plants. New Phytol. 2014, 203, 717–734. [CrossRef][PubMed] 34. Liao, T.; Yuan, D.Y.; Zou, F.; Gao, C.; Yang, Y.; Zhang, L.; Tan, X.F. Self-sterility in Camellia oleifera may be due to the prezygotic late-acting self-incompatibility. PLoS ONE 2014, 9, e99639. [CrossRef][PubMed] 35. Darwin, C. The Effects of Cross and Self-Fertilization in the Vegetable Kingdom; John Murray: London, UK, 1876. 36. Fetscher, A.E. Resolution of male-female conflict in a hermaphroditic flower. Proc. Biol. Sci. 2001, 268, 525. [CrossRef][PubMed] 37. Barrett, S.C.H. Sexual interference of the floral kind. Heredity 2002, 88, 154. [CrossRef][PubMed] 38. Gan, X.; Cao, L.; Zhang, X.; Li, H. Floral biology, breeding system and pollination ecology of an endangered tree Tetracentron sinense oliv. (Trochodendraceae). Bot. Stud. 2013, 54, 55–60. [CrossRef][PubMed] 39. Ashman, T.L.; Morgan, M.T. Explaining phenotypic selection on plant attractive characters: Male function, gender balance or ecological context? Proc. R. Soc. B Biol. Sci. 2004, 271, 553–559. [CrossRef][PubMed] 40. Richards, A.J. The Pollination of Flowers by Insects; Academic Press for the Linnean Society of London: London, UK, 1978; pp. 119–128. 41. Thomson, J.D.; Goodell, K. Pollen removal and deposition by honeybee and bumblebee visitors to apple and almond flowers. J. Appl. Ecol. 2001, 38, 1032–1044. [CrossRef] 42. Thomson, J.D. Pollen transport and deposition by bumble bees in erythronium: Influences of floral nectar and bee grooming. J. Ecol. 1986, 74, 329–341. [CrossRef] 43. Snow, A.A.; Roubik, D.W. Pollen deposition and removal by bees visiting two tree species in Panama. Biotropica 1987, 19, 57–63. [CrossRef] 44. Suzuki, K.; Dohzono, I.; Hiei, K.; Fukuda, Y. Pollination effectiveness of three bumblebee species on flowers of Hosta sieboldiana (Liliaceae) and its relation to floral structure and pollinator sizes. Plant Species Biol. 2002, 17, 139–146. [CrossRef]

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