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1 Feeding the enemy: Loss of nectar and nectaries to herbivores reduces tepal
2 damage and increases pollinator attraction in Iris bulleyana
3
4
5 Ya-Ru Zhu1, Min Yang1, Jana C. Vamosi2, W. Scott Armbruster3,4, Tao Wan5 and
6 Yan-Bing Gong1
7 1 State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University,
8 Wuhan 430072, China
9 2 Department of Biological Sciences, University of Calgary, Calgary T2N1N4, Canada
10 3School of Biological Sciences, University of Portsmouth, Portsmouth PO1 2DY,
11 United Kingdom
12 4Institute of Arctic Biology, University of Alaska, Fairbanks, AK 99775 USA
13 5Key Laboratory of Southern Subtropical Plant Diversity, Fairylake Botanical Garden,
14 Shenzhen 518004, China
15
16
17 Author for correspondence: Yan-Bing Gong
18 e-mail: [email protected] 19 Floral nectar usually functions as a pollinator reward, yet it may also attract herbivores.
20 However, the effects of herbivore consumption of nectar or nectaries on pollination
21 have rarely been tested. We investigated Iris bulleyana, an alpine plant that has showy
22 tepals and abundant nectar in the Hengduan Mountains of SW China. In this region
23 flowers are visited mainly by pollen-collecting pollinators and nectarivorous herbivores.
24 We test the hypothesis that, in I. bulleyana, sacrificing nectar and nectaries to
25 herbivores protects tepals and thus enhances pollinator attraction. We compared rates
26 of pollination and herbivory on different floral tissues in plants with flowers protected
27 from nectar and nectary consumption with rates in unprotected control plants. We
28 found that nectar and nectaries suffered more herbivore damage than did tepals in
29 natural conditions. However, the amount of tepal damage was significantly greater in
30 the flowers with protected nectaries than in the controls; this resulted in significant
31 differences in pollinator visitation rates. These results provide the first evidence that
32 floral nectar and nectaries may be ‘sacrificed’ to herbivores, leading to reduced damage
33 to other floral tissues that are more important for reproduction.
34 Key-words: Iris bulleyana, floral herbivory, nectar and nectary, pollination, sacrificial
35 structures, visual cues
36
37 1. Background
38 Nectar produced by floral nectaries usually serves as a pollinator reward, yet the nectar
39 or nectaries may also attract herbivores. For example, nectar-feeding hawkmoths often
40 act as pollinating herbivores: while adults pollinate solanaceous plants, the larvae
41 consume plant tissues [1]. Some adult insects with chewing mouthparts can directly
42 consume both nectar and plant tissues [2]. We call these "nectarivorous herbivores" and
43 suggest that they may have more complicated effects on plant reproductive success.
1 44 Like nectar thieves and nectar robbers [3-5], nectarivorous herbivores can consume
45 nectar resources and/or damage nectaries, leading to changes in pollinator behavior or
46 even pollinator identity [1]. Nectarivorous herbivores can also affect pollination by
47 damaging floral tissues, e.g., petals or tepals taht serve as effective visual cues for
48 pollinator attraction [6-8]. Sometimes interactions are less costly, or perhaps even
49 positive. For example, the loss of calorie-rich nectar and nectaries to herbivores may
50 result in less damage to reproductively more important but nutrient-poor tepals. These
51 interactions are more likely to occur in the nectariferous Iris species, for which the
52 showy tepals and abundant nectar are, respectively, the main pollinator attractants and
53 rewards [8-10]. However, due to the traditional separation of studies on plant-herbivore
54 and plant-pollinator interactions [11], the influence of nectarivorous herbivory on plant
55 pollination has not yet, to our knowledge, been addressed. Here we present
56 experimental evidence showing that the presence of nectar and nectaries influences
57 herbivore and pollinator behaviors simultaneously in the alpine geophyte Iris
58 bulleyana.
59
60 2. Material and methods
61 Study species and floral damage evaluation
62 A natural population of Iris bulleyana Dykes was studied in a wet meadow in
63 Shangri-La Alpine Botanical Garden (27°54'10"N, 99°38'11"E, 3300 m a.s.l.) in the
64 Hengduan Mountains, southwest China. This species needs pollinators for successful
65 fertilization (Chun-Feng Yang, personal communication). We randomly selected 108
66 flowers in late anthesis and evaluated the type and incidences of herbivore damage on
67 the nectaries (not tepals too?) and/or styles in natural conditions during July 2014
68 (figure 1a,b).
2 69 Flower manipulations and insect observations
70 In addition to the observational survey, we also bagged 30 floral buds on 30 widely
71 spaced plants with nylon-mesh nets to exclude both herbivores and pollinators. Ten of
72 these were set as natural controls (figure 1c,d) and the remaining 20 flowers were
73 included in the protected treatment, which was achieved by wrapping the perianth tube
74 with cello-tape to deter herbivores from eating nectar and nectaries (figure 1e,f).
75 Bagged flowers that had just opened were uncovered and chosen randomly to record
76 both herbivore and pollinator visitation in 10 min intervals during 11:00-16:10 on July
77 4-6, 2014. For each herbivore visit, we recorded its behavior as feeding on (1) nectar
78 and nectaries, (2) tepals or (3) petaloid style. In total, we conducted 41 and 110
79 observation intervals (i.e. 410 and 1,100 min) for the unmanipulated and protected
80 treatments, respectively. Damaging visits to petaloid styles were observed only four
81 times throughout the study period and were therefore excluded from the final analysis.
82 Data analysis
83 We modeled first the effects of treatment (unmanipulated vs. protected), tissue type
84 (nectar and nectaries vs. tepals) and their interactions on herbivory rate (damaging
85 visits/flower in 10 min). Secondly, we modeled the effect of treatment on pollination
86 rate (pollinator visits/flower in 10 min). Observation intervals and flowers were always
87 included as two nested random effects. Because of the response variables were
88 over-dispersed counts, both models were estimated as negative binomial regressions
89 with a log link function [12], as implemented in the R package “lme4”.
90
91 3. Results
92 In natural conditions, 98.2% (106 flowers) of the 108 I. bulleyana flowers exhibited
93 herbivore damage in late anthesis, of which damage 85.2% (92) was only to nectaries
3 94 (figure 1b), and 12.96% (14) was to both nectaries and styles. Only 1.85% (two) had
95 both intact nectaries and styles.
96 During 1,510 min of insect observation, we found that herbivorous adult sawflies
97 (Tenthredo maculiger) frequently chose natural I. bulleyana flowers as mating sites.
98 During mating, nectar and nectaries of I. bulleyana were consumed mainly by female
99 sawflies. These sawflies were observed to bite holes in the perianth tube and consume
100 all the nectar and nectaries inside, but did little damage to the adjacent style and never
101 contacted the stamen and stigma (figure 1b-d). With respect to pollination, we found
102 that solitary bees (Halictus spp.) and hoverflies (Syrphus spp.) collected or ate pollen
103 and made contact with the stigma frequently, but never entered the perianth tube for
104 nectar (figure 1d). To further confirm the pollination effects, two solitary bees were
105 caught individually when they were visiting I. bulleyana. We found 24 and 27 pollen
106 grains on their bodies, respectively, and these were determined as I. bulleyana based on
107 size and shape in comparison with pollen reference collection.
108 In 41 observation intervals, unmanipulated flowers received 81 pollinator visits (75
109 by solitary bees and 6 by hoverflies), plus 80 damaging visits by sawflies, comprising
110 63, 16 and 1 damaging visits to nectaries, tepals and petaloid styles, respectively. In 110
111 observation intervals, protected flowers received 116 pollinator visits (112 by solitary
112 bees and 4 by hoverflies), plus 154 damaging visits by sawflies, comprising 1, 150 and
113 3 damaging visits to nectaries, tepals and petaloid styles, respectively.
114 Treatment (coefficient = -5.09, P < 0.001), tissue type (coefficient = -1.34, P <
115 0.001) and their interactions (coefficient = 6.34, P < 0.001) were all significant in
116 predicting herbivory rate (figure 2a). The herbivory rate of nectaries/nectar on
117 unmanipulated flowers was significantly higher than the herbivory rate on tepals of
118 unmanipulated flowers. It was also higher than the herbivory rate of nectaries/nectar of
4 119 protected flowers. These results indicate that sawflies preferred feeding on nectar and
120 nectaries over tepals in natural conditions and that the protective treatment was
121 effective. The herbivory rate on tepals of protected flowers was significantly higher
122 than the herbivory rate on tepals of unprotected flowers. This suggests that tepals are
123 secondarily chosen as resources by sawflies when nectar and nectaries are unavailable.
124 Finally, the protection treatment resulted in a significant decrease in pollinator
125 visitation rate (coefficient = -0.68, P = 0.003; figure 2b).
126
127 4. Discussion
128 There has been recent renewed interest in the interplay between reproduction and
129 defense. Such interactions between pollination and herbivory systems make it even
130 more challenging to our understanding of the function of floral traits [13,14]. For
131 example, if, in this study, we had regarded floral nectar as only a pollinator reward,
132 neglecting its effects on herbivores, we would have concluded that the nectar of Iris
133 bulleyana had no current function because pollinators collected only pollen. However,
134 our joint assessment of pollination and herbivory demonstrated that nectar and
135 nectaries of I. bulleyana instead play a role in distracting herbivores away from the
136 showy tepals.
137 This novel interpretation of the function of nectar and nectaries is further supported by
138 the results of experimentally protecting the nectaries: this led to an increase in tepal
139 damage and a decrease in pollinator visitation. These results are consistent with the
140 interpretation that floral tissues with higher reproductive importance are essentially
141 protected through the presence of ‘sacrificial’ structures. To some extent, the sacrificial
142 nectar and nectaries of I. bulleyana may play a similar role as the feedings anthers of
143 Melastoma flowers, which attract and reward pollinators, thereby reducing the waste of
5 144 pollen gains of the reproductively more important pollinating anthers [15,16].
145 Of course, the ‘protection’ of tepals by nectar secretion and loss of nectary tissue
146 could just represent an ecological anachronism [17], reflecting simply an incidental
147 consequence of herbivore behavior in the local absence of the ‘proper’, nectar-feeding
148 pollinator. Bumblebees [18,19] and sawflies [20] are both widely distributed in our
149 study area but their interactions with I. bulleyana may vary across years and
150 populations because of ecological context variation [19,21]. The high herbivory rate of
151 nectaries/nectar in this population and study period may also cause the inhibition of
152 visits by nectar-feeding bumblebees, intensifying the anachronistic function of nectar
153 and nectaries.
154 With regard to attracting pollinators to Iris flowers, there is general agreement that
155 visual cues, in the form of extremely large flowers and/or bright colours are vitally
156 important [8,10]. Previous community studies in the same study site have found that
157 both flower size and color can affect the floral choices of different pollinator groups
158 [18,19]. More specific studies further demonstrate that large and showy floral tissues
159 such as bracts can serve as effective visual cues for pollinator attraction [6,7], which
160 might also be true for the conspicuous I. bulleyana with its large floral size in our study
161 (figure 1). Indeed, we found pollinator visitation rate decreased significantly with the
162 increasing tepal damage by herbivores, showing the importance of tepals for the
163 successful pollinator attraction of I. bulleyana. However, there are other explanations
164 that may account for this finding. For example, herbivores may induce/increase
165 volatiles released by nectar and nectaries to enhance the attracting of pollinators and the
166 distracting of the herbivores from the tepals [1,22]. Thus, despite one study showing
167 that visual cues rather than olfactory signals played significant pollinator-attracting
168 roles in their Iris system [10], lack of data on floral volatiles in our study is a limitation.
6 169 Further work should quantify visual cues and floral volatiles of I. bulleyana and explore
170 their effects on pollinator and herbivore behaviors.
171 Overall, nectarivorous herbivory may be common in natural systems because nectar
172 and nectaries are rich in complex nutrients, and adult herbivores have high energy
173 demands. In the situation of severe nectarivory by herbivores, nectar and nectaries may
174 play little or no role in pollination, but instead function as sacrificial resources for
175 antagonists. Thus, nectar and nectaries should be studied in the context of both
176 mutualistic and antagonistic interactions to broaden our understanding of their
177 ecological functions.
178
179 Data accessibility. All data associated with this project are archived in the Dryad
180 Digital Repository at: http://datadryad.org/review?doi=doi:10.5061/dryad.25c4r.
181 Author contributions. YBG, YRZ and MY designed and performed the research,
182 YBG analyzed the data, YBG, YRZ, JCV, WSA and TW wrote the manuscript; all
183 authors agree to be held accountable for the content therein and approve the final
184 version of the manuscript.
185 Competing interests. We have no competing interests.
186 Funding. This work was supported by the NCFC (31100276), HBSF (2011CDA095)
187 and the SZSF (JSGG20140515164852417 & 201519).
188 Acknowledgements. We thank L.K. Dong and M.C. Wei for insect identification, Z.D.
189 Fang and staff from SABG for logistical support, S.M. Vamosi, L.D. Harder, C. Dai, F.
190 Liu, the editors and reviewers for helpful comments.
191
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244 Figure legends
245 Figure 1. Flowers, herbivores and pollinator of Iris bulleyana in unmanipulated (a-d)
246 and protected (e-f) treatments. (a) nectar secreted from nectaries on the inner walls of
247 the perianth tube; (b) flower in late anthesis with intact style but depleted nectar and
248 nectaries; (c) sawfly (Tenthredo maculiger) consuming nectar and nectaries; (d)
249 solitary bee (Halictus sp.) collecting pollen and sawfly consuming nectar and nectaries
250 simultaneously; (e) sawfly attempting to consume nectar and nectaries but deterred by
251 sellotape; (f) sawflies consuming tepals.
252
253 Figure 2. Comparisons of (a) herbivore damage rates on nectar and nectaries vs. tepals,
254 and (b) pollinator visitation rate between unmanipulated and protected treatments on
255 Iris bulleyana. All statistics are significant. See text for details. Bars, mean ± SE.
10