Carotenoids are responsible for the yellow and red color of flowers and these compounds were changing in the flowering of the P. calyculatus. On the initial stages of the bud and anthesis, the flowers showed a yellow color on D0–D2, but on D3 the flowers presented a bright orange color and D4 they turned into a light red color. We can observe that on D2– D3 occur a transition change of yellow light to orange bright color and the flower become an orange intense as the amount of pigment increases; however, our carotenoids quantification did not fit with these flower color changes, only β-carotene the main pigment was constant in their concentration. Carotenoids are a large family of pigments, and are responsible for many of the brilliant red, orange, and yellow color in flowers (Delgado-Vargas et al., 2000). In addition, flowers can contain at least other important group of pigments such as anthocyanins, these are involved in the red color of the flowers (Miller et al., 2011), and we do not discard that these pigments are involved in the flower color transition of light to orange the flower of P. calyculatus. Azpeitia and Lara (2006) found similar flowering color pattern to our results, however, pigment petals did not were analyzed at the different floral stages. Color from floral parts constitutes the major visual attractants for pollinators (Ram and Mathur, 1984). When P. calyculatus flowers change in color after opening, the nature and biogenesis of floral carotenoids and their quantitative concentrations differ at different flower stages. The carotenoid contents that we found were lutein, lycopene, and β-carotene, lutein was the less concentrated at all flower stages; lycopene increased their concentration from D1 to D3 and diminished on D4. A similar behavior was observed for β-carotene, with a high concentration on D0, but a lower content of this pigment on D4. These patterns of carotenoids degradation can be due to the effect of pollinated flower (Ohmiya, 2013). Ram and Mathur (1984) evaluated the color changes subsequent to the anthesis and determined that the pollination was a key factor as a trigger for a rapid carotenoids and anthocyanin synthesis in the flower lifetime; for this reason, we can observed a carotenoid diminished in the lifetime of P. calyculatus flowers. Hummingbirds use their vision principally in finding yellow or bright red color flowers with copious quantities of FN (Miller et al., 2011). Schemske and Bradshaw (1999) found a weak correlation between hummingbird and bee visitors and the color of Monkey flowers (Mimulus lewissi and M. cardinalis.). In this research, hummingbirds did not exhibited preferences by flowers with similar concentrations of carotenoids or anthocyanins, and petal carotenoids significantly decrease the bee visitations, without effect on hummingbirds, concluding that the high concentration of these pigments function primarily to discourage bee visitation. However, in our field's results we quantified a high percentage of bee visitation, these could be due to the effect of different VOCs that attract these insects. The ability of hummingbirds to quickly find rich nectar sources and to return to them suggests that hummingbirds are capable of exerting strong selection on the nectar rewards of flowers (Schemske and Bradshaw, 1999). These results are consistent with our findings due to the fact that hummingbirds do not have an innate preference for P. calyculatus yellow or red flowers and their attraction can be due to the high volumes of nectar in these flowers.

#### Flower Visitors on the Flower Lifetime

Mistletoes of the Loranthaceae family are strongly associated with ornithophilia syndrome due to the flower morphology (tubular structures, colorful, robust corollas, and resistant sexual organs) (Faegri and Van der Pijl, 2013). We found that the P. calyculatus flowers received four types of floral visitors, hummingbirds, butterflies, bees and wasps. Hummingbirds were the main visitors, followed by bees, butterflies, and wasps. The foraging events of hummingbirds begin in the early hours of the morning and these are gradually reduced at midday; however, we found interestingly that as the visits of hummingbirds decreased, the visits of the insects increased, this could be to avoid conflicts of negative interactions between the nectar

consumption. In addition, those foraging visits decreased in the different phenological flower stages and could be a direct effect of the nectar cease. Previous studies carried out on P. calyculatus found that its flowers are visited by four species of hummingbirds, observing a greater presence of visits also in the mornings and decreasing at the midday (Azpeitia and Lara, 2006) similar to our observations. The presence of hummingbirds as main pollinators in other mistletoe species such as P. schiedeanus (Ramírez and Ornelas, 2010), P. robustus (Guerra et al., 2014), and P. auriculatus (Pérez-Crespo et al., 2016) has been observed and therefore they have been cataloged as the main pollinators of these mistletoes, since they describe the hummingbird as an effective carrier of pollen (López de Buen and Ornelas, 1999). The hummingbirds' head dimensions make them ideal for touching the anthers that contain the pollen, they carried the pollen on the head thus when visiting another flower, they can fertilize it. On the other hand, insects have a tiny size and they do not have effective contact with the anthers and pollination is not performed (Azpeitia and Lara, 2006). The presence of hummingbirds is related to the extended phenological flowering is seen as a pattern in mistletoes pollinated by these birds (Galetto et al., 1990; Rivera et al., 1996). It has been observed in different mistletoes that having high nectar production and flower longevity rates suggest a greater attraction of pollinators (Ramírez and Ornelas, 2010). The number of pollinator visits can be influenced by a large number of factors such as environmental conditions (temperature and humidity), attractions (visual and volatiles), and rewards (nectar and pollen) signals (Proctor et al., 1996). The plant ensures the attraction of pollinators through the anthesis of the flower by secreting large amounts of nectar rich in components such as sugars and amino acids that pollinator organisms seek to maintain their biological daily activities (Guerra et al., 2014). In that aspect, the hummingbird is possibly attracted by the sweet taste of the nectar derived from sucrose mainly, as well as by proline and oxo-proline.

In sum, the flowers of P. calyculatus contain different traits such as nectar production enriched with an excellent quality of

#### REFERENCES

biomolecules such as sucrose, glucose, and fructose, and energetic amino acids such as proline and oxo-proline as a reward for pollinators. The quality of sugar is due to the CWIN activity that biochemically is synchronized with the nectar secretion on the flower lifetime, the lower emission of VOCs was involved in the insect attraction, antimicrobial effects, or insect repellence, the showy color flower is composed by different carotenoids with functions of attraction and repellence. This is the first time that different traits are evaluated in this mistletoe flowers. These important suits can enforce pollination specificity and manipulate the behavior to improve the pollination in flowers of P. calyculatus.

#### AUTHOR CONTRIBUTIONS

EQ-R and DO-T designed the study, performed the experiments, and wrote the manuscript. AR-R and ER-C performed the experiments. XC-C performed the statistical tests. JM-T and JE-C provided equipment. MH provided intellectual suggestions.

### FUNDING

This research was supported by the incentive of National Research System (SNI), and supported by the project Bioturbosin Cluster (208090) both of Consejo Nacional de Ciencia y Tecnología (CONACYT) and Secretaria de Energia (SENER).

#### ACKNOWLEDGMENTS

We would like to thank Edith Díaz and Johnattan Hernández for criticism in earlier versions of this manuscript. We also thank Antonio Cisneros for taking the photographs of flower phenology and, CINVESTAV-Irapuato for providing all field facilities.

exclusively hummingbird-pollinated. Ecology 99, 1239–1241. doi: 10.1002/ecy. 2140

Zimmerman, M. (1988). "Nectar production, flowering phenology, and strategies for pollination," in Plant Reproductive Ecology: Patterns and Strategies, Vol. 41, eds J. Lovett Doust and L. Lovett Doust (New York, NY: Oxford University Press), 157–178.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Quintana-Rodríguez, Ramírez-Rodríguez, Ramírez-Chávez, Molina-Torres, Camacho-Coronel, Esparza-Claudio, Heil and Orona-Tamayo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Reduced Responsiveness to Volatile Signals Creates a Modular Reward Provisioning in an Obligate Food-for-Protection Mutualism

Omar F. Hernández-Zepeda, Rosario Razo-Belman and Martin Heil\*

Departamento de Ingeniería Genética, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional-Irapuato, Guanajuato, Mexico

#### *Edited by:*

Clay Carter, University of Minnesota Twin Cities, United States

#### *Reviewed by:*

Bertrand Schatz, Centre National de la Recherche Scientifique (CNRS), France Michael Staab, Albert Ludwigs Universität Freiburg, Germany

> *\*Correspondence:* Martin Heil<EMAIL_ADDRESS>
#### *Specialty section:*

This article was submitted to Functional Plant Ecology, a section of the journal Frontiers in Plant Science

*Received:* 30 March 2018 *Accepted:* 03 July 2018 *Published:* 24 July 2018

#### *Citation:*

Hernández-Zepeda OF, Razo-Belman R and Heil M (2018) Reduced Responsiveness to Volatile Signals Creates a Modular Reward Provisioning in an Obligate Food-for-Protection Mutualism. Front. Plant Sci. 9:1076. doi: 10.3389/fpls.2018.01076 Plants in more than 100 families secrete extrafloral nectar (EFN) to establish food-for-protection mutualisms with ants. Facultative ant-plants secrete EFN as a jasmonic acid (JA)-dependent response to attract generalist ants. In contrast, obligate ant-plants like the Central American "Swollen-Thorn Acacias" are colonized by specialized ants, although an individual host can carry ant colonies from different species that differ in the degree of protection they provide. We hypothesized that hosts that associate simultaneously with various partners should produce rewards in a modular manner to preferentially reward high quality partners. To test this hypothesis, we applied JA to distinct leaves and quantified cell wall invertase activity (CWIN; a regulator of nectar secretion) and EFN secretion by these "local" (i.e., treated) and the "systemic" (i.e., non-treated) leaves of the same branch. Both CWIN activity and EFN secretion increased in local and systemic leaves of the facultative ant-plant Acacia cochliacantha, but only in the local leaves of the obligate ant-plant, A. cornigera. The systemic EFN secretion in A. cochliacantha was associated with an enhanced emission of volatile organic compounds (VOCs). Such VOCs function as "external signals" that control systemic defense responses in diverse plant species. Indeed, the headspace of JA-treated branches of A. cochliacantha induced EFN secretion in both plant species, whereas the headspace of A. cornigera caused no detectable induction effect. Analyses of the headspace using GC-MS identified six VOCs in the headspace of A. cochliacantha that were not emitted by A. cornigera. Among these VOCs, β-caryophyllene and (cis)-hexenyl isovalerate have already been reported in other plant species to induce defense traits, including EFN secretion. Our observations underline the importance of VOCs as systemic within-plant signals and show that the modular rewarding in A. cornigera is likely to result from a reduced emission of the systemic signal, rather than from a reduced responsiveness to the signal. We suggest that modular rewarding allows hosts to restrict the metabolic investment to specific partners and to efficiently sanction potential exploiters.

Keywords: exclusive rewards, extrafloral nectar, obligate mutualism, sanctions, systemic signaling, volatile signals

## INTRODUCTION

Most mutualisms are formed by hosts that interact with multiple partners. Partners can differ in their quality as mutualists, and non-reciprocating partners infer a cost to their host without providing the corresponding benefit (Sachs, 2015). Therefore, theory predicts the evolution of "host sanctions" or other mechanisms that allow hosts to adjust reward provisioning to the quality of the service they receive (Bshary and Grutter, 2002; Kiers et al., 2003; Kiers and Denison, 2008). Host sanctions have been reported for mutualisms such as the legume–rhizobia mutualism, in which plants were reported to "penalize" non Nfixing nodules (Kiers et al., 2003; Westhoek et al., 2017), or for the fig–fig wasp mutualism, in which the fig tree aborted figs that were colonized by non-pollinating wasps (Jandér et al., 2012). Intriguingly, Kiers et al. (2003) and Jandér et al. (2012) observed sanctions to occur in a modular manner: only nodules that did not fix nitrogen or figs that were carrying non-cooperative wasps were sanctioned. Evidently, a modular provisioning of rewards is adaptive in symbiotic systems in which different parts of the same host are colonized simultaneously by different partners. In contrast, hosts that engage in facultative mutualisms with nonsymbiotic partners should provide rewards in a more systemic way, in order to enhance their attractiveness to mutualists that eventually visit the host (Agrawal and Rutter, 1998).

A modular reward provisioning has been reported from symbiotic mutualisms whereas systemic reward production characterizes common facultative mutualisms, but the molecular pathways remain poorly understood that enable plants to allocate rewards in different spatial patterns. Here, we use extrafloral nectar (EFN) secretion to identify the mechanism that controls the modular versus a more systemic production of a reward in an ant-plant mutualism. Extrafloral nectar (EFN) is produced by plants from more than 4,000 species in ca. 750 genera (Weber and Keeler, 2013) to attract ants and other predators, or even parasitoids, all of which act as an indirect defense against herbivores (Heil, 2015). The main components of EFN are mono- and disaccharides and amino acids, but proteins are also frequently reported (Escalante-Pérez and Heil, 2012). The content of metabolically costly compounds and the observation that EFN secretion can be limited by light availability (Bixenmann et al., 2011; Jones and Koptur, 2015) indicate that EFN is a costly reward whose production should be under strict control by the plant. Most EFN-secreting plant species function as facultative ant plants, i.e., they secrete EFN in response to herbivory as a jasmonic acid (JA)-dependent defense mechanism to establish facultative mutualisms with generalist ants, which are attracted from the vicinity (Heil et al., 2001; Roy et al., 2017). The inducibility of EFN secretion by JA finds its mechanistic explanation in the fact that cell-wall invertase (CWIN), which represents a central limiting step in the secretion of nectar (Ruhlmann et al., 2010; Lin et al., 2014; Roy et al., 2017), is induced by JA (Millán-Cañongo et al., 2014). In contrast, socalled myrmecophytes, which have been described from over 100 genera of plants, provide nesting space–and usually also a food reward such as EFN - to colonies of symbiotic "plant-ants". These interactions are considered obligate mutualisms, because the plant-ants depend on their host for food and nesting space, whereas the plants depend on the ants for protection (Heil and McKey, 2003). In simple words, facultative ant-plants recruit generalist ants from the vicinity when defense is actually required, whereas obligate ant-plants provide nesting space and food to a "standing army" of specialized ants (**Figure 1**).

In the present study, we used two EFN-secreting plant species from the same genus to test the hypothesis that the obligate ant-plant provides this reward in a modular way whereas the facultative ant-plant provides the reward systemically, and to identify the molecular mechanism that allows for a modular versus systemic reward production. Obligate ant-plants such as the Mesoamerican "Swollen-thorn Acacias" (sensu Janzen, 1974), such as A. cornigera, A. hindsiii and A. collinsii, offer EFN and cellular food bodies as food rewards for obligate plant-ants from the Pseudomyrmex ferrugineus group (Janzen, 1966; Ward, 1993; Seigler and Ebinger, 1995; Ward and Branstetter, 2017). These ants colonize their host partly, or completely, and protect the colonized parts from herbivores, climbers and pathogens (Janzen, 1967, 1969; González-Teuber et al., 2014; see **Figure 2**, and Supplementary Video File 1). In contrast, other species such as A. farnesiana, A. cochliacantha and A. macracantha (Seigler and Ebinger, 1988) engage in facultative mutualisms with generalist ants species that patrol these plants (Bentley, 1977; Tilman, 1978; Koptur, 1992; Agrawal, 1998). In the latter three plant species, EFN secretion has already been shown to be induced by damage or the exogenous application of JA (Heil et al., 2004). Besides herbivory, mechanical damage, or treatment with JA, EFN secretion can also be induced by volatile organic compounds (VOCs), at least in lima bean (Phaseolus lunatus) (Kost and Heil, 2006; Heil and Silva- Bueno, 2007). In fact, VOCs have been reported as external signals that orchestrate systemic responses to local attack in diverse plant species, comprising both monocots and dicots (Frost et al., 2008; Heil and Ton, 2008; Heil and Karban, 2010; Schrader et al., 2017). Considering this role of VOCs, we hypothesized that a modular versus systemic production of an inducible reward like EFN could result from differences in the emission of plant VOCs or in the responsiveness of the plant to these VOCs (**Figure 1**). In short, in this study, we employ a comparative approach to investigate whether plant VOCs can act as airborne plant hormones that generate different spatial patterns in the EFN secretion by a facultative and an obligate ant-plant.

### MATERIALS AND METHODS

### Plant Species and Study Site

The plant species used in this study were Acacia cornigera (L.) Willdenow, an obligate ant-plant, and Acacia cochliacantha Humb. Bonpl. ex Willd., a facultative ant-plant. All plants selected for this study were shrubs 1–2.5 m tall growing at their natural site in in the coastal area in Southern Mexico close to Puerto Escondido, Oaxaca (∼15◦ 55′N and 097◦ 09′W). Plant species were determined following Janzen, 1974 and Seigler and Ebinger (1988, 1995), and ant species were determined following (Ward, 1993; Ward and Branstetter, 2017), and confirmed by P.S. Ward. Due to the polyphyly of the former genus Acacia s.l.

to local damage. A. cornigera is colonized by a standing army of specialist ants (P. ferrugineus) even in the absence of damage ("healthy", upper panel). These ants are quickly recruited when the plant is locally damaged (lower panel), a response that can be elicited by plant VOCs (Agrawal, 1998; Inui and Itioka, 2007; Mayer et al., 2008; Schatz et al., 2009) or by a strongly modular induction of extrafloral nectar (EFN) secretion (this study). In contrast, A. cochliacantha responds to damage with the attraction of generalist ants from the vicinity, a situation in which the number of ants recruited should correlate positively with the amount of EFN secreted. Since VOCs can control systemic plant responses (Heil and Ton, 2008), a modular versus systemic reward production by the obligate vs. the facultative ant-plant might be explained by differences in the emission of—or the response to—such VOCs.

it has been recommended to term the Mesoamerican clade of the former genus Acacia "Vachellia" (Orchard and Maslin, 2003; The Legume Phylogeny Working Group, 2017), a suggestion that has been discussed intensively (Luckow et al., 2005; Smith and Figueiredo, 2011; Kyalangalilwa et al., 2013). For the sake of reproducibility and in order to allow comparisons with published work, we respect the iconic term "Swollen thorn Acacias" as introduced by Janzen in 1974 and, hereinafter, use the species names as defined in the beforementioned taxonomic keys, which have been used to identify our study species.

### Effect of JA and Ants on EFN Secretion

Earlier observations suggested that obligate Acacia ant-plants secrete EFN constitutively (Heil et al., 2004). However, only plants that were colonized by the obligate plant-ant, P. ferrugineus, had been used in that study, which tempted us to hypothesize a role of the ants in EFN secretion. In order to investigate the effects of ants and of exogenous JA application on EFN secretion, we selected each eight plants of A. cornigera and of A. cochliacantha; all A. cornigera plants were inhabited naturally by P. ferrugineus ants, whereas the A. cochliacantha plants were visited by generalist ants such as Camponotus

branches (white arrows) whereas the remaining parts of the plant remains free of ants and, thus, without a protection from herbivores and pathogens (red arrows). This photo is a part of Supplementary Video File 1. See video file for close-ups.

truncatus, Crematogaster larrea, Pseudomyrmex gracilis (species kindly determined by P.S. Ward). From each plant, we selected three branches that were similar in terms of leaf number and age of the branch, and free of visible damage. All branches possessed at least ten healthy leaves, which were numbered according to their age (leaf 1 being the youngest one, **Figure 3**). One branch per plant served as positive control, i.e., it remained inhabited by P. ferrugineus ants (A. cornigera) or with continuous access for generalist ants (A. cochliacantha). The other two branches were deprived of ants as described earlier (Heil et al., 2004). In short, all thorns were cut off (A. cornigera only) and all ants were removed from the branch. Then, a ring of Tangle Trap <sup>R</sup> (a sticky resin that prevents ants from passing, The Tangle Foot Company http://www.planetnatural.com/product/ tree-tanglefoot-insect-barrier/) was applied around the base of the branch and finally, the branch was covered with a gauze bag to protect the EFN from flying nectar robbers and the leaves from herbivores (Heil et al., 2004). After 2 days, nectaries on one of these ant-free branches were treated with 20 µL of 1 mM aqueous solution of JA pipetted directly on each nectary or, as a negative control, with 20 µL of Milli-Q <sup>R</sup> water. Then, all three branches were deprived of ants and protected from nectar consumers as described above and in Heil et al. (2004). The volume and concentration (in equivalents of sucrose) of the secreted EFN was quantified 24 h later with microcapillaries and a portable refractometer (Atago <sup>R</sup> hand refractometer) for three leaves per branch. Consecutively, these leaves were collected and dried to express EFN secretion as amounts of soluble solids secreted per g of leaf dry mass and 24 h, as described earlier (Heil et al., 2004).

#### Modular vs. Systemic Response to JA

In order to evaluate whether the two Acacia species investigated here secrete EFN as a modular or a systemic response, we selected eight plants each of A. cornigera and A. cochliacantha as described above and selected two branches per plant, using the same criteria as above but making sure that each branch had at least ten fully expanded, healthy leaves. One branch per plant was treated by applying 20 µL of 1 mM of aqueous JA solution to the nectaries of leaves 1, 5, and 10, whereas 20 µL of Milli-Q <sup>R</sup> water was applied to the same nectaries on the control branches. After 24 h, EFN secretion was quantified as described above. However, in this experiment, EFN secretion was quantified individually for each leaf, including both the "local" (i.e., treated) as well as the "systemic" (i.e., non-treated) leaves of the same branch.

In order to quantify CWIN activity, an independent set of eight plants from each species were selected and treated as described above. The nectary tissue was collected 1 h after JAtreatment and stored immediately on dry ice in 1.5 mL Eppendorf tubes. Samples were transferred to a portable Deep Freezer (https://www.thomassci.com/Equipment/General-Purpose-Refrigeration/\_/25L-Super-Low-Temperature-Portable-Deep-Freezer?=&q=Ultra+Low+Freezer) and stored at −40◦C until

use. The activity of CWIN was quantified according to Millán-Cañongo et al. (2014) and Ruhlmann et al. (2010) with some modifications. Briefly, 0.10 g of frozen tissue was ground and mixed with 500 µL cold 50 mM HEPES/NaOH (pH 8.0, containing 5 mM MgCl2, 2 mM EDTA, 1 mM MnCl<sup>2</sup> and 1 mM CaCl2). Samples were incubated on ice for 10 min and then centrifuged at 13,000 g for 10 min at 4◦C. The pellets that contained the cell walls with associated invertases were washed three times with 500 µL extraction buffer by resuspension and centrifugation as described above, each time discarding the supernatant. Then, the pellets were washed three times with 500 µL of 80 mM sodium acetate (pH 4.8). Then, 300 µL of 80 mM sodium acetate (pH 4.8) were added to the pellets, suspended, and the mixture was incubated at 37◦C. Every 5 min, an aliquot of 20 µL was taken and mixed with 200 µL of hexokinase (HK) reaction solution ("glucose (HK) assay kit"; Sigma-Aldrich, http://www.sigmaaldrich.com). After reaching steady state, 100 µL of an aqueous 100 mM solution of sucrose was added, and the absorption was measured at 340 nm in a lQuant <sup>R</sup> Spectrophotometer, ThermoSpectronic, microplate reader every 5 min for 40 min with Gen 5 software (Biotek https://www.biotek.com/products/software-robotics-software/ gen5-microplate-reader-and-imager-software/).

the airflow toward the younger leaves (C) or away from the plant (D). See materials and methods section for details.

### Effect of VOCs on EFN Secretion

In order to investigate a putative role of VOCs on EFN secretion, we adapted the experimental design from (Heil and Silva- Bueno, 2007) in which the youngest leaves of a branch served as local "receiver" leaves to which air flow from the headspace of other leaves on the same branch was experimentally manipulated (**Figure 3**). In short, four plants each of A. cornigera and A. cochliacantha were selected as described above. Five branches that possessed at least six healthy leaves (of which the youngest leaves 1-3 served as receivers) were selected on each plant and were deprived of ants as described above. Then, the branches were subjected to one of the following treatments (see **Figure 3** for details). In the first treatment (**Figure 3C**), three mature leaves (4-6) were treated by spraying a 1 mM aqueous JA solution until the surfaces of all leaves were covered, allowed to dry, and then bagged in plastic foil ("Bratschlauch", Toppits, Minden, Germany; a PET foil that does not emit detectable amounts of volatiles). One side of the bag was left open and an open-flow system was created by placing a plastic tube (30 × 2 cm; inner surface lined with Bratschlauch) on the opposite side, creating a continuous air flow placing a ventilator (video card cooler "Evercool EC-4010," Steren, Mexico City, Mexico, supplied with 4.5 V) at the upper end of the tube. Then, air flow from the treated leaves was directed toward the three youngest leaves (1-3) on the same branch. In the second treatment (**Figure 3**, Panel D), the air flow from the treated leaves was directed away from the branch, leaving leaves 1-3 exposed to ambient air. As a control, we manipulated air flow was as in the first treatment, but leaves 4-6 were treated with water as a control. Ultimately, two branches of the same plant were exposed to air coming either from a JA-treated or a Milli-Q <sup>R</sup> water-treated branch of the other study plant species (i.e., leaves of A. cornigera were exposed to air coming from A. cochliacantha or viceversa).

### Collection and Analysis of VOCs

In order to compare the VOC profiles of A. cornigera and A. cochliacantha plants, two branches of each ten plants per species were selected and the youngest 10 leaves on one branch per plant were spray-treated with 1 mM of aqueous JA solution as described above, whereas the youngest ten leaves on the other branch were spray-treated with water as a control. After allowing leaves to dry, the branches were bagged in Bratschlauch. VOCs were collected over 24 h in a closed-loop system as described in Donath and Boland (1995), using micro-pumps (model DC 06/21 FK, Fürgut, Tannheim, Germany) and filters (1.5 mg of charcoal, CLSA- Filters, Le Ruissaeu de Montbrun, France). The VOCs were desorbed from the filters using 40 µL of dichloromethane with 1- bromodecane (98%, Aldrich) at a concentration of 100 ng µL −1 as an internal standard, and samples were injected directly into a gas chromatograph-electron impact ionization mass spectrometer (GC- EIMS) system (Agilent 7890 series gas chromatograph interfaced to an Agilent 5975 electron impact ionization mass-selective triple axis detector; Agilent Technologies Santa Clara, CA, USA). The separation was performed using a HP5- FAPP column (30 m long, 0.32 mm internal diameter and 0.5 mm film thickness) under the following conditions. Injector temperature 180◦C, detector temperature 230◦C, initial temperature 70◦C, then ramped up at 5◦C min−<sup>1</sup> to 120◦C, then ramped at 8◦C min−<sup>1</sup> to a final temperature of 210◦C, which was maintained for 12 min. The mass spectra were analyzed with MassHunter 2017 by Agilent Technologies <sup>R</sup> , and compounds were preliminarily annotated with "NIST MS Search Program v.2.0g," Library version 11, and AMDIS version 2.71 from Agilent Technologies <sup>R</sup> , and then confirmed with authentic standards purchased from Sigma-Aldrich and Fluka Chemie (now Merck, purchased via Sigma-Aldrich, Toluca, Mexico).

### Statistical Analysis

The all data obtained were subjected to t-student tests or ANOVA with posthoc Tukey-HSD. For the statistical analyses we used the program R <sup>R</sup> version 3.3.0 (R studio).

### RESULTS

### Effects of JA on EFN Secretion and CWIN Activity

Plants of A. cornigera secreted significantly more EFN on antinhabited and JA-treated ant-free branches as compared to watertreated ant-free branches (p < 0.001 for the comparisons ants vs. ant-free and JA vs. ant-free; p > 0.05 for ants vs. JA; see **Figure 4A**). In contrast, in the case of A. cochliacantha, only JA treatment had a significant effect on EFN secretion (p < 0.001; see **Figure 4B**), whereas no significant difference could be detected between ant-free branches and branches to which ants had access (p > 0.05, see **Figure 4B**). The differences between both species became even more pronounced when we investigated the systemic effects of local JA application (**Figure 5**). In the case of A. cornigera, application of JA to the nectaries on leaves 1, 5, and 10 significantly induced EFN secretion in the directly treated nectaries (difference between treated and control leaves: p < 0.001 at leaf positions 1, 5, and 10), whereas the EFN

FIGURE 4 | Exogenous JA induces EFN secretion in A. cornigera and A. cochliacantha. Extrafloral nectar (EFN) secretion on ant-free branches of (A) A. cornigera and (B) A. cochliacantha responded to exogenous JA (black bars) as compared to controls (light gray bars). EFN secretion was also high on A. cornigera branches that were colonized by mutualistic (P. ferrugineus) ants but not on A. cochliacantha branches that were visited by generalist ants (dark gray bars). Bars represent means ± SD of EFN secretion rates in mg of sucrose equivalents per g of leaf dry mass and 24 h, different letters above bars indicate significant differences (p < 0.001, according to ANOVA followed by Tukey HSD, n = 8).

secretion rates on the systemic leaves of the treated branches showed no significant difference to the secretion rates on the corresponding leaves on untreated branches (p > 0.05 at leaf positions 2, 3, 4, 6, 7, 8, and 9; see **Figure 5A**). In contrast, all leaves on the treated branches of A. cochliacantha responded with a significant increase in EFN secretion to JA application to the nectaries on leaves 1, 5, and 10, independently whether they were "local" (i.e., directly treated) or "systemic" leaves (difference between leaves on treated and control branches: p < 0.001 at all 10 leaf positions, see **Figure 5B**).

The same patterns were observed in CWIN activity. In A. cornigera, CWIN activity in nectary tissue responded significantly (p < 0.001) to the direct JA application to nectaries

on leaves 1, 5, and 10, but showed no significant differences between nectaries on leaves 2, 3, 4, 6, 7, 8, and 9 on JA-treated versus control branches (p > 0.05) (**Figure 6A**). In the case of A. cochliacantha, however, CWIN activity was significantly higher in all leaves on the treated branches as compared to the corresponding leaves on control branches (p < 0.001 at all 10 leaf positions), and no differences could be detected among the "local" and the "systemic" leaves on the treated branches (**Figure 6B**).

### VOCs From *A. cochliacantha* Induce EFN Secretion in Both Species

When we treated mature leaves (4-6) of A. cornigera with JA to study the putative role of JA-responsive VOCs, the EFN secretion by these leaves was induced, as shown by the

significant differences (p < 0.001) between EFN secretion rates observed on JA-treated leaves versus controls (**Figure 7A**). In contrast, the EFN secretion on the young leaves (1–3) of the same branches did not respond significantly to exogenous JA applied to the mature leaves, independently of whether the young leaves were exposed to the headspace of the treated, mature leaves (p < 0.05) or not (**Figure 7A,** treatments I and II). However, EFN secretion on young leaves (1–3) of A. cornigera was significantly induced after the exposure to the headspace of JA-treated leaves of A. cochliacantha (p < 0.001, see **Figure 7A**, treatment IVa). Correspondingly, EFN secretion on mature leaves of A. cochliacantha responded significantly to exogenous JA (**Figure 7B**), and the EFN secretion on young leaves was induced by the headspace of JA-treated mature leaves: that is, EFN secretion on leaves 1-3 was significantly

branches of A. cornigera (A) and A. cochliacantha (B) branches is depicted for young leaves (no. 1–3) and mature leaves (4–6) that were treated with JA or exposed to the headspace of differently treated leaves. Treatment I, mature leaves treated with JA, young leaves exposed to air from mature (i.e., induced) leaves. Treatment II, mature leaves treated with JA, young leaves exposed to environmental air. Treatment III, mature leaves treated with Milli-Q® water, young leaves exposed to air from mature (i.e., non-induced) leaves. Treatment IV, response of young leaves (1–3) to the headspace from heterospecific leaves (i.e., A. cornigera exposed to headspace of A. cochliacantha and vice-versa). The heterospecific emitter branches were either treated with JA (IVa) or with Milli-Q® water (control, IVb). Bars represent means ± SD of EFN secretion rates in µg per g leaf dry mass and 24 h, different letters above bars indicate significant differences (p < 0.001 according to ANOVA followed by Tukey HSD, n = 4).

higher (p < 0.001) on leaves that had been exposed to the headspace coming from JA-treated mature leaves (**Figure 7B**, treatment I) as compared to ambient air or the headspace of water-treated control leaves (**Figure 7B**, treatments II and III). Finally, no significant effect (p > 0.05) on EFN secretion by A. cochliacantha could be detected for the headspace of A. cornigera, independently of whether the A. cornigera branch had been treated with JA or not (**Figure 7B,** treatment IVa,b).

#### The Voc Blends of *A. cornigera* and *A. cochliacantha* Are Different

Both species responded to exogenous JA with the induced emission of various VOCs (**Table 1**). We could detect

The headspace was sampled over 24 h after the treatment and compounds identified in *Figure 8* as numbered peaks are listed according to their retention time (RT), values represent mean peak areas (×10<sup>7</sup> ) ± SD per g of dry mass of the emitting leaves of n = 10 independent samples per species and treatment, ND, Not detected. All compounds were confirmed by co-injection with commercial standards.

six different compounds in the headspace of JA-treated A. cornigera branches, among which the monoterpene alcohols, α-terpineol and β-linalool, and 2,6-dimethyl-1,3,5,7-octatetraene ("dimethyl-octatetraene" in **Figure 8A**), were released only from JA-treated plants (**Figure 8A**). All six compounds that we detected in the headspace of JA-treated A. cornigera branches could also be detected in the headspace of JAtreated A. cochliacantha branches. However, A. cochliacantha emitted six additional compounds: cis-hexenyl isovalerate, the monoterpene, α-cubebene, and the sesquiterpenes longicyclene, germacrene, β-caryophyllene and α-farnesene (**Figure 8B**). According to t-tests performed separately for each VOC, JA treatment induced the emission of four out of six compounds significantly (p < 0.005) in case of A. cornigera and of all 12 compounds in case of A. cochliacantha (**Table 1**, **Figures 8A,B**).

### DISCUSSION

In the present study, we show that EFN secretion by the obligate ant-plant, A. cornigera, responded in a modular manner to local JA-treatment, whereas in the facultative ant-plant, A. cochliacantha, EFN secretion responded more systemically. The activity of CWIN in the tissue of the individual nectaries closely resembled the patterns seen in EFN secretion rates in both species, confirming earlier reports that EFN secretion is controlled at the level of individual nectaries (Orona-Tamayo et al., 2013). In A. cornigera, an enhanced EFN secretion also correlated with the colonization by resident mutualistic P. ferrugineus ants, whereas generalist ants visiting the nectaries of A. cochliacantha did not exert any detectable EFN-inducing effect. A modular sanctioning of non-reciprocating symbionts has been reported from different types of mutualisms. However, the signals that allow for different spatial patterns in the production of rewards remain to be identified. Thus, the observation of different spatial patterns in the reward production by two closely related species made our system highly suitable to search for the causal mechanism that controls these patterns.

### EFN Secretion in Both Species Responds to *A. cochliacantha* VOCs

Interestingly, the headspace of JA-treated leaves of A. cochliacantha induced EFN secretion in the systemic leaves of the same branch. As reported earlier, inducible VOCs can act as airborne "external" signals that control systemic responses to local damage (Heil and Ton, 2008; Scala et al., 2013; Loreto et al., 2014). In contrast, EFN secretion by systemic leaves of A. cornigera did not respond to the headspace of the JA-treated leaves of the same branch. This observation shows that in A. cornigera, VOCs do not serve as a systemic, EFN-inducing signal, and raised the question whether the two species studied here differ in the emission of VOCs, or in the responsiveness of EFN secretion to these VOCs. Indeed, the headspace of JA-induced A. cochliacantha branches readily induced EFN secretion in A. cornigera. Although an exposure of A. cornigera leaves to VOCs from A. cochliacantha is not likely to resemble a natural situation of ecological relevance, this finding clearly demonstrates that EFN secretion by A. cornigera, in principle, can be induced by exogenous volatile signals.

#### VOCs as Plant-to-Ant Signals

Which ones among the VOCs that were emitted from A. cochliacantha were responsible for the EFN-inducing effect? The role of VOCs in the signaling from host plants to their ants has been studied in various ant-plant systems. In Acacia spp. and Macaranga spp., VOCs emitted from facultative and obligate ant-plants were compared to identify compounds that

might serve as host-finding cues for foundresses (Jurgens et al., 2006; Razo-Belman et al., 2018). Agrawal (1998) was the first to focus on the defensive aspect and studied plant-ants as a VOC-responsive mechanism of protection. Inui and Itioka (2007) found that diverse species of Macaranga myrmecophytes emitted different VOC profiles when damaged and that the aggressiveness of resident Crematogaster ants toward damaged leaves also differed among the host species. Using leaf pieces from different Piper species, Mayer et al. (2008) found that the resident ants responded more strongly to damaged leaves from obligate than from facultative Piper ant-plants, whereas Schatz et al. (2009) used the same approach to show that obligate plant-ants responded to leaf pieces of their host plant more strongly than a non-defending exploiter ant (Schatz et al., 2009). Curiously, this behavior could be elicited using, among other VOCs, pure hexanal (Schatz et al., 2009), a green leaf volatile already reported by Agrawal (1998) to elicit practically the same response as plant sap in Azteka plant-ants. That is, ubiquitous plant VOCs that are emitted from most plants upon damage, or even infection (Heil, 2014; Quintana-Rodriguez et al., 2015, 2018), might be the triggers that ants use to detect host plant damage. More importantly, all the beforementioned six studies focused on the direct chemical communication from the plant to the ants, whereas in the present study, we focused on the VOC-mediated signaling within the plant.

#### Plant VOCs as Defence-Inducing Hormones

The headspace of A. cornigera caused no detectable induction of EFN secretion, which makes it reasonable to assume that the active VOCs can be found under those compounds that were emitted only, or in much higher amounts, by A. cochliacantha. Defence-inducing effects are commonly reported for green leaf volatiles such as (E)-2-hexenal or (Z)-3-hexenyl acetate (Scala et al., 2013; von Mérey et al., 2013; Loreto et al., 2014; Sharma et al., 2017). For example, cis-hexenyl isovalerate has been reported to induce EFN secretion in Phaseolus lunatus (Heil et al., 2008) and indeed, this compound could be detected only in the headspace of JA-treated A. cochliacantha branches. We could not detect other green leaf volatiles in our analyses, which might be partly due to the particular difficulties to collect small, highly volatile compounds under field conditions or to the detection threshold of our GC-MS analyses. Nevertheless, our control samples were practically free of detectable VOCs and all six VOCs that we detected in the headspace of A. cornigera had already been reported from this plant species (Razo-Belman et al., 2018). These facts make us confident that our results adequately resemble the major VOCs that are emitted from our study species. Five of the six compounds that were exclusive for A. cochliacantha were mono- or sesquiterpenes, a group of VOCs for which defense-inducing effects are much less frequently reported than for green leaf volatiles (Sharma et al., 2017). However, an artificial blend consisting of R - (–) linalool, β-caryophyllene, methyl salicylate, cis-jasmone, (cis)-3 hexenyl acetate, β-ocimene, (3E)-4,8-dimethylnona-1,3,7-triene (DMNT) and (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) induced EFN secretion in lima bean (Phaseolus lunatus) (Kost and Heil, 2006), DMNT and TMTT induced pathogenesisrelated (PR) genes and lipoxygenase (a central step in the synthesis of JA) in P. lunatus, and β-ocimene induced PR genes in the same species (Arimura et al., 2000). Likewise, a mixture of α- and β-pinene induced PR1 gene expression in Arabidopsis (Riedlmeier et al., 2017), whereas β-caryophyllene triggered membrane depolarization, which is a very early step in plant defense induction, in Solanum lycopersicon (Zebelo et al., 2012). Among these compounds, β-caryophyllene and β-ocimene were the quantitatively dominant compounds in the headspace of JA-treated A. cochliacantha branches, and β-caryophyllene was exclusive to this species. Taken together, our results make it highly likely that VOCs that are emitted only – or in much higher amounts–from A. cochliacantha leaves function as a systemic EFN-inducing signal, and that the strictly modular response in EFN secretion that we observed in A cornigera is caused by a reduced emission of these volatile signals, rather than a reduced responsiveness to the signals.

### Optimized Rewarding by Modular vs. Systemic Responses

Modular responses in plant defense have been suggested to be driven by herbivores in order to optimize host sharing (Lee et al., 2017). However, in the case of our study system, it appears more likely that local EFN secretion enables A. cornigera to focus reward production on specific parts of the plant surface. EFN is a costly reward (Escalante-Pérez and Heil, 2012) and can be a limiting factor for ant colony growth (Byk and Del-Claro, 2011). In the case of Swollen-thorn Acacias, higher EFN secretion rates can shift the competitive balance between defending mutualist ants and non-defending exploiters to the benefit of the mutualists (Heil, 2013). The reduced emission of VOCs by A. cornigera is likely to represent a consequence of the frequently proposed reduction of direct defense traits in obligate ant-plants (Janzen, 1966; Rehr et al., 1973; Koricheva and Romero, 2012), rather than a specific adaptation to avoid a systemic induction of EFN secretion.

We also hypothesized that systemic EFN secretion enables a facultative ant- plant to attract more ants and gain a better defensive service when it is required. Although being reasonable (Agrawal and Rutter, 1998), surprisingly little evidence has been reported to support this assumption. In fact, the defensive effects of EFN secretion are highly context-dependent (Heil, 2015; Jones et al., 2017) and EFN secretion can even be counterproductive if ants start to exclude other, more efficient defenders Koptur et al., 2015). Inducing EFN secretion with exogenous JA increased the number of defending ants and decreased the number of herbivores showing up on Macaranga tanarius plants (Heil et al., 2001), and similar patterns were found on P. lunatus tendrils that were exposed to VOCs or treated with JA to enhance EFN secretion (Kost and Heil, 2008). However, all leaves had been treated in these studies, making a separation of local and systemic effects impossible. A study at the ecosystem level showed that ant abundance increased with higher EFN secretion rates and presented evidence for a strong competition among the ants for this valuable food reward (Lange et al., 2017). That ants compete for EFN has been reported from different systems (Blüthgen and Fiedler, 2004; Xu and Chen, 2010; Lange et al., 2017), which makes it likely that, under most circumstances, enhanced amounts of EFN that are secreted on larger areas of a facultative ant- plant should enhance the number of attracted ants (**Figure 1**).

## CONCLUSIONS

In the case of an obligate ant-plant, a modular rewarding of resident ants (**Figure 2**, and Supplementary Video 1) should allow for an optimized investment in protection, because these ants defend only a restricted part of the plant. In contrast, the protection of a facultative ant-plant should increase when higher numbers of visiting ants are recruited via an enhanced investment in reward production. Interestingly, volatile signals represent a molecular mechanism that controls systemic responses to local events and that can generate marked differences in the provisioning of rewards among closely related plant species that engage in different types of food-for-protection mutualisms.

### AUTHOR CONTRIBUTIONS

OH-Z and RR-B designed and performed the field experiments, analyzed the data and wrote a first version of the manuscript. MH helped with the design of the experiments and data analyses and all authors worked on the final version of the manuscript.

#### ACKNOWLEDGMENTS

We thank Rosa María Adame-Álvarez and Enrique Ramírez-Chávez for invaluable help with the volatile analyses, Octavio Martínez de la Vega for help with the statistical design and

#### REFERENCES

Enrique Hurtado Bautista for help with scientific illustrations. Philip S. Ward kindly confirmed the identity of the ant species and Madgalena Ríos generously provided Supplementary Video 1. CONACyT de México is acknowledged for financial support (grant no. 301808 to OH-Z, 237052 to RR-B, and 251102 to MH).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018. 01076/full#supplementary-material

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Hernández-Zepeda, Razo-Belman and Heil. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Ant-Pollinator Conflict Results in Pollinator Deterrence but no Nectar Trade-Offs

#### Nora Villamil <sup>1</sup> \*, Karina Boege<sup>2</sup> and Graham N. Stone<sup>1</sup>

<sup>1</sup> Ashworth Laboratories, Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, United Kingdom, <sup>2</sup> Instituto de Ecología, Departamento de Ecología Evolutiva, Universidad Nacional Autónoma de México, Mexico City, Mexico

Direct and indirect negative interactions between ant guards and pollinators on ant-plants are expected for two reasons. First, aggressive ants may deter pollinators directly. Second, pollinators benefit from plant investment in reproduction whilst ants benefit from plant investment in indirect defense, and resource allocation trade-offs between these functions could lead to indirect conflict. We explored the potential for ant-pollinator conflict in a Mexican myrmecophile, Turnera velutina, which rewards ants with extrafloral nectar and pollinators with floral nectar. We characterized the daily timing of ant and pollinator activity on the plant and used experiments to test for direct and indirect conflict between these two groups of mutualists. We tested for direct conflict by quantifying pollinator responses to flowers containing dead specimens of aggressive ant species, relative to unoccupied control flowers. We assessed indirect conflict by testing for the existence of a trade-off in sugar allocation between ant and pollinator rewards, evidenced by an increase in floral nectar secretion when extrafloral nectar secretion was prevented. Secretion of floral and extrafloral nectar, activity of ants and pollinators, and pollen deposition all overlapped in daily time and peaked within the first 2 h after flowers opened. We found evidence of direct conflict, in that presence of ants inside the flowers altered pollinator behavior and reduced visit duration, although visit frequency was unchanged. We found no evidence for indirect conflict, with no significant difference in the volume or sugar content of floral nectar between control plants and those in which extrafloral nectar secretion was prevented. The presence of ants in flowers alters pollinator behavior in ways that are likely to affect pollination dynamics, though there is no apparent tradeoff between plant investment in nectar rewards for pollinators and ant guards. Further studies are required to quantify the effect of the natural abundance of ants in flowers on pollinator behavior, and any associated impacts on plant reproductive success.

Keywords: Turnera velutina, ant-plant, floral nectar, extrafloral nectar, resource allocation, indirect interactions, Myrmecophily

### INTRODUCTION

Extrafloral nectaries, domatia, and food bodies are all means by which ant-plants (comprising myrmecophiles and myrmecophytes) (Rosumek et al., 2009; Del-Claro et al., 2016) attract and support ants by providing nesting sites or nutrients (Rico-Gray and Oliveira, 2007; Rosumek et al., 2009). In return, ants attack herbivores, prune climbing vines and prevent fungal and microbial

#### Edited by:

Massimo Nepi, University of Siena, Italy

#### Reviewed by:

Kleber Del-Claro, Federal University of Uberlandia, Brazil Rubén Torices, Estacion Experimental de Zonas Aridas (EEZA), Spain

> \*Correspondence: Nora Villamil<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Functional Plant Ecology, a section of the journal Frontiers in Plant Science

Received: 20 April 2018 Accepted: 05 July 2018 Published: 14 August 2018

#### Citation:

Villamil N, Boege K and Stone GN (2018) Ant-Pollinator Conflict Results in Pollinator Deterrence but no Nectar Trade-Offs. Front. Plant Sci. 9:1093. doi: 10.3389/fpls.2018.01093 infestation on plant tissues (Bentley, 1977; Rosumek et al., 2009). This mutualistic interaction is termed myrmecophily.

Interactions involving two or more types of mutualists of a single host are common in nature, but multispecies interactions are much less studied than pairwise and intraguild mutualisms (Strauss, 1997; Tscharntke and Hawkins, 2002; Strauss and Irwin, 2004; Adler, 2008; Melián et al., 2009; Koptur et al., 2015). To date, most research on plant-animal interactions has focused on pairwise relationships (e.g., plant-herbivore, plantpollinator, plant-fungus) in isolation from the community in which they are embedded (Strauss, 1997; Herrera, 2000; Dáttilo et al., 2016; Del-Claro et al., 2018). This pairwise approach necessarily oversimplifies reality (Herrera, 2000) since plants interact sequentially or simultaneously with each of pollinators, herbivores, herbivore predators and pathogens (Armbruster, 1997). Furthermore, plant interactions with one partner or guild can also affect relationships with other groups or guilds (Armbruster, 1997) and alter outcomes from mutualistic to antagonistic (Strauss, 1997; Strauss et al., 1999; Herrera, 2000; Strauss and Irwin, 2004; Del-Claro et al., 2016). As a result, a growing number of studies are focusing on multispecies and multitrophic interactions (Melián et al., 2009; Fontaine et al., 2011; Nahas et al., 2012; Pineda et al., 2013; Dáttilo et al., 2016). It might be expected, for example, that the presence of predatory ants can influence pollinators, with top-down effects on plant fitness. This makes ant-plants, which rely on ants for defense against herbivores and on pollinators for seed set, a model tritrophic system in which to explore the dynamics of multispecies and multitrophic interactions.

Here, we focus on disentangling ant-pollinator interactions that occur when both mutualists share a host plant. Previous work has revealed evidence of ant-pollinator conflict in such systems (Yu and Pierce, 1998; Stanton et al., 1999; Gaume et al., 2005; Ness, 2006; Palmer and Brody, 2007; Frederickson, 2009; Stanton and Palmer, 2011; Malé et al., 2012; Assunção et al., 2014; LeVan et al., 2014; Hanna et al., 2015). Antpollinator conflict is expected for two main reasons. Firstly, both mutualists share with their host interest in different plant functions. Pollinators benefit from plant resource allocation to reproduction (i.e., flowers, floral nectar (FN) and pollen), whilst ants benefit from allocation to growth and defense [i.e., vegetative structures bearing extrafloral nectar (EFN) or domatia] (Yu and Pierce, 1998; Frederickson, 2009; Palmer et al., 2010). This could result in a conflict mediated by plant rewards, known as indirect conflict. Floral and extrafloral nectar share sugar as a common currency, providing potential for a trade-off and also a means of quantifying investment in each. Secondly, because ant guards actively defend their host plant as a means of protecting food and/or nesting sites, they may also repel or attack pollinators (Ness, 2006; Stanton and Palmer, 2011; Chamberlain and Rudgers, 2012), and this drawback of ant guards is known as direct conflict.

Castration is an extreme example of direct ant-pollinator conflict in which guarding ants destroy or consume the reproductive meristems, floral buds or flowers of their host plant (Yu and Pierce, 1998; Stanton et al., 1999; Gaume et al., 2005; Palmer and Brody, 2007; Frederickson, 2009; Malé et al., 2012). Such castrating behavior inevitably reduces availability of flowers and hence floral rewards for pollinators. It has been suggested that the ultimate cause of castration by patrolling ants is promoting reallocation of plant resources from reproduction to growth (Yu and Pierce, 1998; Frederickson, 2009; Malé et al., 2012), and hence increases the availability of resources on which ant colonies depend. In ant species that are obligate inhabitants of ant-plants, colony size is limited by the number of domatia (Fonseca, 1993, 1999; Orivel et al., 2011), which is positively correlated with plant investment in growth. Consequently, resource allocation strategies toward these two mutualists should be approached in a linked way because plant investment toward growth may come at the cost of investment to reproduction, and vice versa. And so, plant investment in rewards for each mutualist reward may be affected, either positively via linkage, or negatively via trade-offs. Furthermore, because plants interact with both mutualists simultaneously the presence of one mutualist may increase or decrease presence of the other.

Even in those species that do not castrate their host, ants' aggressive behaviors might threaten and deter pollinators, compromising plant reproduction (Ness, 2006; Assunção et al., 2014; LeVan et al., 2014). Avoidance of such direct conflict has been suggested to explain plant architecture or behaviors that reduce spatial (Raine et al., 2002; Malé et al., 2015; Martínez-Bauer et al., 2015) or temporal overlap of ant guards and pollinators (Gaume and Mckey, 1999; Gaume et al., 2005; Nicklen and Wagner, 2006; Ohm and Miller, 2014; Malé et al., 2015), and the presence of ant-repelling compounds which exclude ants from flowers when pollen is released (Junker et al., 2007; Willmer et al., 2009; Ballantyne and Willmer, 2012).

Extrafloral nectar is a key resource mediating multispecies interactions in many plant communities, and plants bearing extrafloral nectaries comprise up to a third of species in some biomes (Dyer and Phyllis, 2002; Rudgers and Gardener, 2004; Davidson and Cook, 2008; Dyer, 2008), particularly in tropical dry forests, savannas and cerrados (Rico-Gray and Oliveira, 2007; Assunção et al., 2014). The importance of ant-plants as food resources for mutualists in a given plant community is enhanced if these plants also secrete FN and pollen for pollinators. Management of ant-pollinator conflict in such a way that the crucial services provided by both mutualist groups are maintained is thus likely to be part of the adaptive landscape of many plant species. Ant-plants with extrafloral and floral nectaries represent an excellent system in which to test for trade-offs in resource allocation, competition amongst mutualistic guilds, and assess whether plant strategies minimize direct and indirect conflicts between their mutualists. To our knowledge, no study has addressed both direct and indirect ant-pollinator conflict in a single ant-plant system.

Here we tested for direct and indirect ant-pollinator conflict on a Mexican endemic ant-plant, Turnera velutina. In particular, we assessed whether Turnera velutina reduces the potential for conflict through the daily timing of FN and EFN release. We also tested for potential indirect (nectar-mediated) and direct (deterrence) conflicts between ants and pollinators. We addressed the following specific questions: (i) What are the daily timings of nectar reward secretion, ant activity, and floral visitation? (ii) Does the presence of patrolling ants deter pollinators from the flowers? (iii) Do ant species vary in their deterrence for pollinators? (iv) Are T. velutina plants able to reallocate extrafloral nectar resources into floral nectar resources (increasing reward availability to flower visitors)?

### MATERIALS AND METHODS

#### Study Site and System

Field experiments were conducted in coastal sand dunes at the CICOLMA Field Station in La Mancha, Veracruz, in the Gulf of Mexico (19◦ 36′ N, 96◦ 22′W, elevation < 100 m). The climate is warm sub-humid, with a rainy season during the summer (June to September), an annual precipitation of 1,100–1,500 mm, and a mean annual temperature ranging between 24 and 26◦C (Travieso-Bello and Campos, 2006). Experiments were carried out in November 2014 at four sites with high densities of Turnera velutina (Passifloraceae). Greenhouse experiments were conducted in a shade house at CICOLMA.

Turnera velutina is an endemic perennial shrub (Arbo, 2005) and myrmecophile (Cuautle and Rico-Gray, 2003). At La Mancha, T. velutina is patrolled by at least seven ant species (Cuautle et al., 2005; Zedillo-Avelleyra, 2017) and its main herbivores are the specialist caterpillars of the butterfly Euptoieta hegesia (Nymphalidae). Extrafloral nectar is provided in paired cup-shaped glands located at the bottom of the leaf blade at the junction with the leaf petiole, on the underside of the leaves (**Figure 1A**, Elias et al., 1975; Villamil et al., 2013). Although it flowers year-round, flowering peaks during summer (Cuautle et al., 2005). Flowers last one day, are animal-pollinated (Sosenski et al., 2016) and provide FN at the base of the corolla (**Figure 1**). Honeybees (Apis mellifera) are the dominant pollinators at La Mancha, accounting for 94% of the visits (Sosenski et al., 2016) and collect both pollen and floral nectar, but the role of other floral visitors is yet to be investigated as effective pollinators. There is no spatial segregation of patrolling ants and floral visitors in Turnera velutina since flower buds emerge from the axillary meristems of leaves bearing extrafloral nectaries (Villamil, 2017). Furthermore, EFN volume and sugar content are higher at the flower stage than for either buds or fruits (Villamil, 2017).

#### Fieldwork Methods

#### Mutualist Activity Curves: Patrolling Ants and Pollinators

We quantified daily activity of patrolling ants and flower visitors on T. velutina by surveying flowers (n = 120 plants, n = 1,604 flowers) and their associated leaves at all four La Mancha sites in November 2014. We observed all open flowers for 2 min every hour throughout anthesis (0800–1300 h), with one observer at each site. Every hour, we counted the total number of floral visitors and ants patrolling extrafloral nectaries across all flowers within a site. We sampled the same sites over multiple days. Since these are one-day flowers, we considered each site-day as a replicate (n = 10 site-days; 43.23 ± 2.89 flowers/site-day), and

FIGURE 1 | Flower of Turnera velutina (top) with arrows indicating the location of the floral nectaries at the base of the corolla, between the petals, and leaves (bottom) with arrows indicating the location of extrafloral nectaries, on the underside of the leaves.

incorporated site-and-day effects into our statistical modeling (see below).

#### Nectar Secretion and Pollen Deposition Curve

Nectar secretion and pollen deposition data were collected from 4 sites within CICOLMA over 5 consecutive days in September 2015. We visited a single site per day (with one exception which was visited twice) and sampled FN and EFN secretion rate and pollen deposition from 1 flower per plant for 5 plants per site (n = 20 plants). Flowers were sampled every hour during the anthesis period (0800–1300 h). Flowers were bagged with tulle bags before anthesis and FN and EFN were collected every hour during anthesis. The first collection was taken as soon as the corolla was fully open. Flowers were re-bagged between measurements to avoid nectar consumption and a masking tape band with Tanglefoot was applied on the stem below the flower to exclude ants for the duration of the experiment. FN was extracted using a 1 µl capillary inserted into each of the five nectaries in a single flower (Minicaps Disposable capillaries, Hirschmann Laborgerate, In 20◦C, ISO 7550, R<0.5%, CV<1.0%, Germany). EFN was also collected from the glands using 1 µl capillaries. Nectar volume was measured using a digital caliper (Mitutoyo Digimatic) and sugar concentration was determined in ◦Brix (g sucrose per 100 g solution) using a 0–50◦B hand held refractometer (Reichert, Munich, Germany). To obtain all of the sugar from extrafloral nectaries, the glands were washed with 2 µl of deionized water using a 0–5 µl micropipette and the sugar concentration of the wash was quantified using the refractometer. Total sugar content in FN and EFN was calculated from volume and ◦Brix values according to Comba et al. (1999) using the formula:

$$s = d\nu \text{C/100}$$

where s is the sugar content (µg), v is the nectar or wash volume (µl), and d is the density of a sucrose solution at a concentration C (g of sucrose per 100 g solution) as read on the refractometer. The density was obtained according to Comba et al. (1999) using the formula:

$$d = 0.0000178C^2 + 0.003791C + 0.9988603$$

A different flower from each of these twenty plants was chosen and marked, but never bagged. We collected one stigma every hour, to sample the pollen deposited 1 h, 2 h, and 3 h postanthesis. A small proportion of flowers contained a fourth stigma, contributing to a small dataset for 4 h post anthesis. Stigmas were stored individually in labeled Eppendorf tubes. Stigmas were individually mounted on slides for fresh squash glycerin preparations. Slides were sealed using nail polish and kept in a fresh and dry environment at 22◦C. Each slide was labeled with the site, day, hour, and plant identity. The number of pollen grains on each stigma was counted under a microscope, and changes in numbers at each time interval plotted to generate the pollen deposition curve.

#### Direct Conflict

To test whether non-ant flower visitors detect and avoid flowers with ants, visitation was observed in flowers with and without dead ants for 4 flowers on each of 40 plants (n = 160 flowers). Plants > 80 cm in height were haphazardly selected and flowers bagged at 0700 before anthesis. Once the corollas had opened, three ant corpses from a single ant species (either Dorymyrmex bicolor, Brachymyrmex sp., or Paratrechina longicornis) were placed inside each of three flowers/plant. A fourth flower was left as an ant-free control. Ant corpses were placed on the inner surface of the petals in each flower. These species were chosen because they were amongst the most abundant species (see Results), displayed patrolling behaviors on T. velutina, and were detected as potentially differing in their effects on Apis mellifera pollinators by a previous study (Villamil et al. unpublished data). Additional flowers were removed from the plant to standarise floral display across all individuals. Ants were killed in 50% ethanol, which was allowed to evaporate for 30 min at ambient temperature (28–35◦C) from all samples to prevent ethanol vapors from influencing pollinator behavior. Flowers containing dead ants were observed for 20 min, recording pollinator identity, visit frequency and duration, and associated pollinator behaviors. Observations were conducted during October-November 2016 with four simultaneous observers collecting data from different plants.

When assessing the effects of ants inside the flowers on pollinator visitation we only considered visits by Apis mellifera, since this is the dominant T. velutina pollinator in this population (Sosenski et al., 2016) and accounted for 91% of the visits in this experiment. We classified honeybee behaviors as "inspection" or "contact." Inspection behaviors comprised either approaching or hovering over a particular flower without landing. Contact behaviors were those that occurred inside the flower, between landing and take-off, and comprised foraging on pollen or nectar resources or standing on the petals, anthers or stigmas.

#### Indirect Conflict

To test possible trade-offs in plant resources between FN and EFN we conducted a greenhouse experiment on 72 plants from 18 different maternal lines (2–4 siblings per maternal family, generated from field individuals). Plants were kept under greenhouse conditions in a shade house located within CICOLMA field station. Plants were grown in 1L plastic pots with a substrate of local soil and vermiculite (50:50) and watered every other day (for rearing details see: Ochoa-López et al., 2015). During the experiment, plants were watered every night, and extrafloral nectaries were sprayed with water to wash away any nectar secretion from previous days and to prevent fungal infections on the glands. Pre-anthesis buds were bagged either the night before or during the morning before anthesis to prevent any nectar theft by unexpected insects that may occasionally enter the shade house.

Pairs of maternal siblings were chosen and randomly assigned to either control or experimental groups. The extrafloral nectaries in all leaves of experimental plants were clogged by applying Mylin transparent textile paint in the nectary cup. Extrafloral nectaries from control plants were left intact (unclogged) and droplets of the same paint were applied on the leaf blade above the glands to match any unintended effects on plants across treatments. Very young floral buds were marked, and their extrafloral nectaries clogged from their emergence, throughout their development, and until anthesis. The droplets on the extrafloral nectaries or leaf blades were checked daily and replenished when required, especially when a new leaf emerged in order to guarantee uniform and continuous application of the clogging treatment across all leaves.

FN secretion was measured before the clogging treatment was applied, and once again after the young clogged/marked buds became flowers. The aim was to test whether flowers that were unable to secrete EFN invested more sugar resources in floral nectar. FN was collected from control and treatment flowers between 1300 and 1500 h using a 1 µl microcapillary pipette. Nectar volume and total sugar mass was estimated and calculated as described above.

#### Statistical Methods

All statistical analyses were conducted in R version 3.23 (R Core Team 2016). All mixed effects models were fitted using the 'multcomp' R package (Bates et al., 2016) and post hoc Tukey comparisons were fitted using the 'multcomp' R package (Hothorn et al., 2008), unless stated otherwise.

#### Mutualist Activity and Reward Secretion Curves

To test whether ant or pollinator activity changed over daily time, we fitted a Poisson mixed model with either the number of ants patrolling or the number of floral visitors as the response variable. We fitted time of day as a fixed effect, with linear and quadratic terms to detect non-linear activity patterns over time. The number of flowers per site was fitted as a log-transformed offset to control for floral display, since we recorded visitor counts per site rather than counts per individual flower (see fieldwork methods). Flowers of T. velutina last for a single day, and because multiple flowers were sampled on a given site on a given day, we fitted site identity as a random effect to account for differences between site and day variation in variables that could influence ant abundance, such as resource availability, ant diversity, or the abundance/proximity of ant nests. We also included an observation-level random effect (OLRE) where each data point receives a unique level of a random effect to control for overdispersion (Hinde, 1982).

To test if FN and EFN secretion changed over the anthesis period we fitted a Poisson mixed model independently for each nectar type, with sugar mass (µg) as the response variable. Nectar sugar content is usually estimated in µg, and we report our raw data in such units. However, to facilitate model convergence we re-scaled our response variable (sugar content) from µg to g, and rounded it to the next integer to better fit a Poisson distribution. We fitted time of day as a fixed effect, with linear and quadratic terms to detect non-linear activity patterns over time. We fitted plant identity as a random effect, and, included an observation-level random effect.

#### Timing of Daily Activity and Secretion Peaks

We computed the time at which mutualist activity and nectar secretion reached their maximum by calculating the time at which the slope (i.e., the differential of the fitted model with respect to time) for each variable is zero and then solving for hour, as follows:

$$\begin{aligned} \mathcal{V} &= \beta\_{\text{hour}} \ast hour + \beta\_{\text{hour}^2} \ast hour^2\\ \frac{d\mathcal{V}}{d\_{\text{hour}}} &= \beta\_{\text{hour}} + 2\beta\_{\text{hour}^2} \ast hour\\ 0 &= \beta\_{\text{hour}} + 2\beta\_{\text{hour}^2} \ast hour\\ \frac{-\beta\_{\text{hour}}}{2\beta\_{\text{hour}^2}} &= \, hour\\ hour &= -\frac{1}{2} \ast \frac{\beta\_{\text{hour}}}{\beta\_{\text{hour}^2}} \end{aligned}$$

#### Direct Conflict

The effect of different ant species on the visitation frequency was tested using Poisson mixed effects models. We fitted the number of visitors as the response variable, and ant species inside the flower (Control, Dorymyrmex bicolor, Brachymyrmex sp., or Paratrechina longicornis) was fitted as a fixed effect. Because these are 1 day flowers, plant-day identity was chosen as a random effect to control for individual variation in floral and extrafloral nectar investment. We also included an observationlevel random effect. Post hoc Tukey comparisons were used to test differences in visit duration between the four treatments.

We tested whether ant species inside the flower differed in their effect on the likelihood with which a pollinator displayed an inspection behavior using a binomial mixed model. The presence or absence of inspection behaviors was coded as the response variable and ant species was fitted as a fixed effect. As random effects we fitted the plant-day identity, and the visitor identity. For those visits where the pollinator displayed an inspection behavior, we fitted the proportion of time per visit spent displaying inspection behaviors using a Gaussian mixed model with logit transformation for data normality. Ant species was included as a fixed effect, and plant-day identity, and the visitor identity were fitted as random effects.

Finally, differences in the duration of inspection or contact behaviors in flowers containing different ant species were analyzed using Gamma mixed models. Mixed effects models were fitted independently for each behavior, but using the same model structure fitting visit duration per flower as the response variable. Ant species inside the flower was fitted as a fixed effect. Plant-day identity was chosen as a random effect to control for individual variation in floral and extrafloral investment, and daily weather variations. We also included an observation-level random effect. Post hoc Tukey comparisons were used to test differences in visit duration between the four treatments.

#### Indirect Conflict

For each plant, we estimated the difference in FN produced before and after the extrafloral nectaries were clogged as follows: DFN = PostFN − PreFN, where D-FN is the difference, PostFN is the FN production after the extrafloral nectaries secretion was prevented and PreFN is the FN production before the extrafloral nectaries were clogged. Differences in volume and sugar content between control and experimental flowers were tested using mixed effects models. Both variables had normal distributions and so Gaussian mixed effects models were fitted using the same model structure: The clogging treatment was fitted as a fixed effect, and the maternal family was fitted as a random effect to independently explain variation in both nectar volume and sugar content.

#### RESULTS

#### Mutualist Activity, Reward Secretion, and Pollen Deposition Curves

Activity curves show that both patrolling ants and floral visitors were most active within the first 2 h post-anthesis (**Figure 2**), although the visitation peak by potential pollinators predicted from model estimates was on average over an hour before the predicted peak for ant activity (9 min post-anthesis for potential pollinators, 90 min post-anthesis for ant patrolling; **Table 1**).

FIGURE 2 | Timing of (A) reward secretion (n = 20 flowers-leaves), (B) mutualist activity (n = 1,604 flowers; n = 10 sites/day) and (C) pollen deposition (n = 20 flowers) in Turnera velutina during the anthesis period (08:30–12:30), showing raw data from field observations (mean ± se). Red circles show floral nectar and the activity of pollinators in flowers, whilst green triangles show extrafloral nectar and the activity of ants at extrafloral nectaries.

Estimates at which the maxima for mutualist activities and reward production are reached are shown in the last three columns. Peak hour is the time in hours estimated, mpa shows minutes post-anthesis when the maxima is reached, and time of day indicates an approximation of when that activity is likely to occur, although this varies depending on the season and the time of sunrise. \*P < 0.05; \*\*\*P < 0.001.

On average, a flower and its associated leaf secreted a total of 2,815 ± 767 µg of sugar via floral and extrafloral nectar throughout the 4.5 h anthesis period. The total sugar content in FN was 149 ± 19.3 µg of sugar, whilst total extrafloral sugar was 2,665 ± 765 µg. Thus, the relative sugar contributions of floral and extrafloral nectar in a leaf-flower module were 5.3 and 94.7%, respectively.

Floral and extrafloral nectaries of T. velutina are both able to quickly replenish nectar after experimental removal by non-destructive sampling of the same flowers over the entire anthesis period (**Figure 2A**). FN and EFN are secreted simultaneously during anthesis and with the highest amount of sugar content secreted during the first 2 h of anthesis (**Figure 2A**), although their secretion peaks predicted from model estimates are slightly mismatched (**Table 1**). EFN secretion peaked 68 min after our first collection, which was taken as soon as flowers were fully open, whilst FN peaked 23 min after the first collection. The timing of peaks in secretion of the two types of nectar matches that for the mutualists that harvest each resource. Peak pollen deposition occurred at the beginning of anthesis and steadily declined over time (**Figure 2C**). Hence, pollen deposition data were analyzed using a linear model without fitting hour as a squared term and we did not estimate timing of daily maxima using model derivations (**Table 1**).

We recorded 1,535 ant visitors from nine ant species patrolling extrafloral nectaries of T. velutina at CICOLMA (**Table 2**). Dorymyrmex bicolor, Paratrechina longicornis, and Brachymyrmex spp. accounted for 68.5% of the total ants observed, and 77.35% of the patrolling ants, after excluding Monomorium spp. that were never observed displaying patrolling behaviors on T. velutina and are mostly parasitic consumers of FN and EFN (lestobiotic) (Ettershank, 1966; Bolton, 1987).

#### Direct Conflict

We recorded 991 floral visitors, of which 907 (91.5%) were by the honeybee Apis mellifera. Of the remaining 8.5%, 61 visits were by native bees, 11 by flies (Diptera), 10 visits by Lepidoptera, one visit by a beetle (Coleoptera) and one by a wasp (Hymenoptera) (**Table 2**).

Neither the presence of ants inside flowers nor their identity had any significant effect on the number of honeybees visiting the flowers (**Figure 3A**, **Table 3**). The presence of the most aggressive ant species, Dorymyrmex bicolor, increased the likelihood of a pollinator displaying inspection behaviors by 20% (**Figure 3B**), and increased by 12% the proportion of time per visit spent displaying inspection behaviors rather than foraging or pollinating the flower (**Figure 3C**). Finally, the presence of Dorymyrmex bicolor and Paratrechina longicornis inside the flowers halved the duration of contact visits compared to control flowers without ants, or to flowers with Brachymyrmex sp. ants inside.

#### Indirect Conflict

Clogging extrafloral nectaries on the leaves associated with newly emerged floral buds had no effect on their FN volume [LRT (1,49) = 0.21; P = 0.64, **Table 3**, **Figure 4B**] or sugar content [LRT (1,49) = 0.087; P = 0.77, **Table 3**, **Figure 4A**]. Differences in FN volume and sugar content (FNpost−treatment − FNpre−treatment) were positive in plants under both control and clogged treatments (**Figure 4**).

### DISCUSSION

Our findings show that both ants and pollinators are active while flowers are open (**Figure 2B**), that FN and EFN are simultaneously secreted (**Figure 2A**), and that pollen deposition occurs when ants are actively patrolling (**Figure 2C**). Consequently, in T. velutina guarding ants and pollinators operate in close spatial and temporal proximity, implying that direct and indirect conflict could occur in this system. We found, however, no evidence for indirect, nectar-mediated, conflict between ants and pollinators, since plants did not reallocate resources toward floral nectar, even when 95% of the sugar investment during anthesis, which is in EFN, is prevented (**Figure 2**). We found evidence for direct conflict, as the presence of dead individuals of patrolling ant species inside flowers was associated with both higher frequency of inspection behaviors in potential pollinators, and reduced visit duration (time spent inside flowers) (**Figure 3**). Taken together, these effects increased handling time per flower and reduced pollinator foraging efficiency. Nonetheless, this result was obtained under an experimental setting using dead ants placed inside flowers. Further studies are required to test (i) the effect of living patrolling ants on pollinator visitation, and (ii) the impact of any such effects on plant fitness. The latter are crucial to understanding whether there is ongoing

TABLE 2 | Abundance and identity of the ants recorded patrolling extrafloral nectaries during the 2014 census and the floral visitors recorded during the direct conflict experiment in 2016 on Turnera velutina plants.

control with no ants; 1P, Paratrechina longicornis; 2M, Brachymyrmex sp.; 3D, Dorymyrmex bicolor ants. (n = 40 plants; n = 160 flowers).

selection on Turnera velutina to manage direct ant-pollinator conflict.

### Mutualist Activity, Reward Secretion and Pollen Deposition

Floral and extrafloral nectar were secreted simultaneously and rapidly replenished in T. velutina, especially during the first 2 h post anthesis (**Figure 2**). Replenishment is a general feature of EFN secretion (Pacini et al., 2003; Pacini and Nepi, 2007; Pacini and Nicolson, 2007). In fact, we are unaware of any report documenting extrafloral nectaries incapable of replenishing secretion after consumption (Pacini et al., 2003; Pacini and Nepi, 2007; Pacini and Nicolson, 2007; Escalante-Pérez and Heil, 2012; Orona-Tamayo et al., 2013; Heil, 2015). In contrast, species vary in whether FN is replenished or not (for details on floral nectar dynamics see: Pacini et al., 2003; Nicolson et al., 2007; Willmer, 2011). We suggest that rapid resupply is crucial in short-lived flowers, such as the one-day flowers of Turnera species, because it makes the flower attractive again for another visit, potentially increasing pollen transfer, pollen deposition, and seed set. Interestingly, Dutton et al. (2016) reported no FN resupply in flowers from three congeners (Turnera ulmifolia,

\*P < 0.05; \*\*\*P < 0.001.

Turnera subulata, and Turnera joelii), when sampling FN in the morning and afternoon, finding no FN secretion in the afternoon collection. These observations show either variation in nectar resupply within closely related species displaying similar floral biology, or perhaps suggest that shorter term dynamics in the nectar supply of the other three species were not detected by the sampling methods used. The latter highlights the need to test floral resupply within short time scales, because over long sampling intervals, flowers can both secrete and reabsorb nectar (Pacini et al., 2003; Nicolson et al., 2007; Willmer, 2011). Finding no standing crop when a flower is resampled in the afternoon does not rule out replenishment after emptying in the morning, and then reabsorption later in the day (Kearns and Inouye, 1993).

Model-based estimation of the daily timing of the maxima of nectar secretion suggest that floral nectar secretion peaks a few minutes after the corolla is fully open (**Table 3**) and 45 min before peak EFN sugar secretion (**Figure 2A**). This represents a slight mismatch in the timing of rewards for ants and pollinators, which may underlie the 85 min mismatch in estimated peaks of ants and floral visitor activity (**Table 1**). To our knowledge, temporal segregation in the activity of ants and pollinators has been reported only for obligate (myrmecophytic) species patrolled or tended by a single ant species at a time (Humboltia brunonis (Fabaceae; Gaume et al., 2005), Hirtella physophora (Chrysobalanaceae; Malé et al., 2015), and Opuntia imbricata (Cactaceae; 2014)). In some specialized systems, ant and pollinator activity occurs in close proximity and simultaneously, but conflict is prevented by ant-repellent floral volatiles [Vachellia zanzibarica (Fabaceae; Willmer and Stone, 1997)] and Vachellia hindsii (Fabaceae; Raine et al., 2002). On the other hand, temporal overlap in ant activity at extrafloral nectaries and pollinator visitation to flowers has been reported for facultative ant-plants associated with many ant species simultaneously [Vachellia constricta (Fabaceae; Nicklen and Wagner, 2006), Acacia myrtifolia, (Acacia sensu stricto, Fabaceae; Martínez-Bauer et al., 2015), and Heteropterys pteropetala (Malpighiaceae; Assunção et al., 2014)]. Our results add Turnera velutina to the list of facultative myrmecophiles with synchronized ant and pollinator activity (**Figure 2**). This synchronous myrmecophile vs. segregated myrmecophyte pattern is consistent with evidence that ants in obligate mutualisms are more aggressive and better defenders (Chamberlain and Holland, 2009; Rosumek et al., 2009; Trager et al., 2010), but may impose greater ecological costs on host pollination. We suggest that temporal segregation of mutualists and/or ant repellent floral volatiles are alternative strategies that reduce such costs. Further studies on the timing of pollinator, ant visitation and ant aggressivity in a wider range of systems are required to test the temporal component of this hypothesis.

### Direct Conflict

We showed that dead ants inside the flowers of T. velutina have an impact on honeybee behavior (**Figure 3**). Ant presence was correlated with shorter honeybee flower visits (**Figure 3C**), an increase in the proportion of visitors displaying inspection behaviors, and increased duration of inspection behaviors per visiting bout (**Figure 3**). We interpret longer inspection behavior to indicate increased caution in the bees (as previously assumed by: Altshuler, 1999; Ness, 2006; Junker et al., 2007; Assunção et al., 2014; Cembrowski et al., 2014). Our findings are consistent with work on Heteropterys pteropetala in which plastic ants inside flowers negatively affected pollination (Assunção et al., 2014). Results for H. pteropetala differ from ours in that the bees pollinating H. pteropetala showed significantly reduced visitation rates to flowers containing plastic ants. In contrast, honeybees in T. velutina did not visit flowers containing ant corpses less frequently than control flowers (**Figure 3A**). In both systems, ants feeding at extrafloral nectaries did not hinder pollination (Assunção et al., 2014; Villamil et al., unpublished data). This suggests that while pollinators avoid ants in flowers, plants may have evolved other mechanisms to prevent ants from entering flowers, resulting in only rare encounters between ants and pollinators.

Although experiments that place ant cues on flowers can tell us about the response of pollinators to ants, they must be interpreted with caution as an indicator of current antpollinator conflict. Firstly, because ants may rarely enter flowers (Villamil et al. submitted). Secondly, by placing such ant cues in flowers we may be violating existing ant-excluding or antrepelling plant mechanisms (Willmer, 2011). Thirdly, in contrast to such experimental treatments, ants do not naturally remain in the flowers for long periods (Assunção et al., 2014), and only a low proportion of flowers may be occupied at any one time. For instance, in T. velutina only 10% of the flowers are occupied by ants (Villamil et al., submitted).

### Does Herbivore Deterrence Match Pollinator Deterrence?

Although some studies have documented variation among ant species in aggression toward herbivores (Ness, 2006; Miller, 2007; Ohm and Miller, 2014), little is known about the effect of different patrolling ant species with varying levels of aggressivity on pollinator visitation (Ness, 2006; Miller, 2007; LeVan et al., 2014; Ohm and Miller, 2014). Nonetheless, a positive correlation between the level of defense provided and the level of pollinator deterrence they exert has often been assumed since ant traits involved in defense (patrolling activity and aggressivity) are likely to be the same as those involved in pollinator deterrence (Ohm and Miller, 2014). Bees tend to forage in a way that maximizes the net benefit of each foraging trip (Stephens and Krebs, 1986; Jones, 2010; Cembrowski et al., 2014). When foraging in ant-plants, this benefit might be maximized if foragers avoid flowers or patches where predation risk is high (Dukas, 2001; Dukas and Morse, 2003; Ness, 2006; Jones and Dornhaus, 2011; Assunção et al., 2014), as could be the case when encountering ant species that attack pollinators. Some ants also consume FN and pollen, and such plants may represent high risk foraging environments with low net rewards for pollinators (Ness, 2006). Shorter or fewer visits to such flowers may be a pollinator strategy to maximize foraging efficiency by avoiding flowers, plants, or patches with high predation risk (Jones and Dornhaus, 2011).

In T. velutina, the most aggressive ant guard, Dorymyrmex bicolor, had the strongest effect on pollinator behavior (**Figure 3**), while Brachymyrmex sp. ants inside the flowers did not reduce the duration of pollinator visits. The least effective anti-herbivore ant species, Paratrechina longicornis (Villamil unpublished data), halved the duration of pollinator visits (**Figure 3**). In Ferocactus wislizeni, plants tended by Solenopsis xyloni, the most aggressive ant species, had fewer and shorter pollinator visits (Ness, 2006). Such differences are consistent with pollinator sensitivity to ant aggressiveness. In contrast, although ant exclusion in Opuntia imbricata significantly increased pollinator visitation, there were no differences in impacts associated with different ant species (Ohm and Miller, 2014), and no evidence that the more aggressive guard (Liometopum apiculatum) had a stronger deterring effect on pollinators (Ohm and Miller, 2014). Whether the level of ant aggressivity toward herbivores correlates positively with the ecological costs on pollination via pollinator deterrence remains unknown (but see: Ness, 2006; Miller, 2007; LeVan et al., 2014; Ohm and Miller, 2014), and should be tested, not assumed.

## Indirect Conflict

Our experimental approach found no evidence for a trade-off in sugar investment in extrafloral and floral nectar in T. velutina. We conclude that there is no indirect nectar-mediated conflict between guarding ants and pollinators in Turnera velutina, and that pollinators do not obtain greater rewards when rewards for patrolling ants are eliminated.

We found only two previous studies testing indirect, nectarmediated ant-pollinator conflict by quantifying sugary rewards (FN and EFN) to both mutualists (Chamberlain and Rudgers, 2012; Dutton et al., 2016). Previous work on other Turnera species by Dutton et al. (2016) found evidence of a tradeoff in two of the three Turnera species tested; removing EFN decreased FN and vice versa in T. ulmifolia and T. subulata, but not in T. joelii. Interestingly, both Turnera species in which trade-offs were detected by Dutton et al. (2016) invested equally in FN and EFN, whilst T. joelli (which showed no trade-off) invested more in EFN (Dutton et al., 2016). The same pattern holds for T. velutina, a species with an asymmetric investment toward EFN, which accounts for 95% of the sugar allocation per leaf-flower module, and where we found no trade-off or resource reallocation from EFN to FN (**Figure 4**). Unfortunately data on FN and EFN volume and sugar content were not reported for the cotton species (Chamberlain and Rudgers, 2012).

One possible reason for lack of a trade-off is that sugar is not a limiting resource for the plant. If so, there would be no reason to expect dynamic reallocation. Estimates of the metabolic costs of nectar secretion vary, and while some studies suggest low metabolic costs (O'Dowd, 1979: EFN accounts for 1% of the total energy invested per leaf), others indicate investment of up to 37% of daily photosynthesis in floral nectar (Southwick, 1984; Pyke, 1991). A second reason, which applies in particular to comparative cross-species analyses rather than experimental manipulations, is that investment in both forms of nectar may be influenced by other aspects of life history strategy. Chamberlain and Rudgers (2012) found no significant negative correlations between extrafloral nectary and floral traits in a comparative analysis across cotton (Gossypium) species, and correlations were significantly positive in 11 of 37 cotton species. Foliar extrafloral nectary volume was positively associated with plant investment in floral nectar, rejecting the hypothesis of trade-offs among investments in pollinators versus bodyguards in Gossypium. Several potential mechanisms underlying the positive correlations between FN and EFN have been proposed, including pleiotropy, and genetic, physiological or ecological linkage (Chamberlain and Rudgers, 2012). The pleiotropy or genetic linkage hypothesis could be tested using genome sequencing (Chamberlain and Rudgers, 2012). Positive correlations could also arise from physiological or ecological linkage. Traits such as FN and EFN may be physiologically linked. However, the fact that in Gossypium FN volume was most strongly correlated with foliar EFN volume, but FN was weakly correlated with bracteal EFN volume (Chamberlain and Rudgers, 2012) questions the physiological linkage hypothesis since bracteal and floral nectaries are spatially closer than floral and extrafloral nectaries, but they are not strongly correlated. We suggest that lack of any trade-off could also indicate that FN and EFN may be phenotypically integrated as a functional module for mutualist attraction. Although formal analyses are required to test this hypothesis, we think it is a strong possibility since T. velutina leaves are phenotypically integrated modules in which leaf economics, defensive and morphological traits covary and are ecologically linked (Damián et al., 2018). Whatever the drivers of these positive correlations may be, available evidence suggests that plants may experience fewer investment trade-offs among different functional traits than previously assumed.

## CONCLUSIONS

To our knowledge, trade-offs between extrafloral and floral nectar traits have been studied in 41 species from two genera: 37 Gossypium species (Chamberlain and Rudgers, 2012), and four Turnera species, including this study (Dutton et al., 2016; Villamil, 2017, **Figure 2**). Negative correlations or evidence for trade-offs have been found in only two of these species: T. ulmifolia and T. subulata (Dutton et al., 2016), representing less than 5% of the species studied. Although many more studies are required to shed light on quantitative trends of floral and extrafloral investment in plants, trade-offs between floral and extrafloral seem infrequent. On the other hand, evidence of direct conflict with patrolling ants reducing pollinator visitation frequency and duration, inducing inspection behaviors and increasing foraging time has been widely reported (Rudgers and Gardener, 2004; Ness, 2006; Chamberlain and Rudgers, 2012; Malé et al., 2012, 2015; Assunção et al., 2014; Koptur et al., 2015; Martínez-Bauer et al., 2015). We suggest that these two issues are not isolated, and hypothesize that positive correlations between FN and EFN investment in ant-plants may be a plant strategy to compensate or lure pollinators to apparently risky flowers.

## DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this manuscript will be made available by the authors upon request.

## AUTHOR CONTRIBUTIONS

NV conceived the ideas, all authors designed the experiments. NV conducted fieldwork, processed, and analyzed the data. NV led the writing of this paper, with critical inputs from KB and GS.

## FUNDING

Thanks to the Mexican taxpayers who funded NV via a CONACYT PhD scholarship. Research costs were funded by Davis Trust Research Grant awarded to NV (2016), Wellcome-BBSRC Insect Pollinator Initiative: Urban Pollinators: their ecology and conservation (BB/I000305/1) awarded to GS, and PAPIIT-UNAM IN211314 grant awarded to KB.

## ACKNOWLEDGMENTS

Thanks to S. Ochoa-López who kindly donated the plants for the greenhouse experiment, X. Damián-Domínguez who provided insightful thoughts for the experimental design and to J. Hadfield who provided valuable inputs toward the statistical analyses. Thanks to the volunteers who helped out during fieldwork: A. Bernal, A. Martínez, R. Ríos, I. Silva, and A. Fournier, to the staff at CICOLMA station, to H. Köck who helped processing pollen samples, and to R. Pérez-Ishiwara for logistic support.

### REFERENCES

by extrafloral nectar: fidelity, cheats, and lies. Insectes Soc. 63, 207–221. doi: 10.1007/s00040-016-0466-2

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Villamil, Boege and Stone. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Crop Domestication Alters Floral Reward Chemistry With Potential Consequences for Pollinator Health

Paul A. Egan<sup>1</sup> \*, Lynn S. Adler<sup>2</sup> , Rebecca E. Irwin<sup>3</sup> , Iain W. Farrell<sup>4</sup> , Evan C. Palmer-Young<sup>2</sup>† and Philip C. Stevenson4,5

<sup>1</sup> Department of Plant Protection Biology, Swedish University of Agricultural Sciences, Alnarp, Sweden, <sup>2</sup> Department of Biology, University of Massachusetts Amherst, Amherst, MA, United States, <sup>3</sup> Department of Applied Ecology, North Carolina State University, Raleigh, NC, United States, <sup>4</sup> Royal Botanic Gardens, Kew, Richmond, United Kingdom, <sup>5</sup> Natural Resources Institute, University of Greenwich, London, United Kingdom

#### Edited by:

Clay Carter, University of Minnesota Twin Cities, United States

#### Reviewed by:

Neelendra K. Joshi, University of Arkansas, United States Kevin Davies, The New Zealand Institute for Plant & Food Research Ltd., New Zealand

> \*Correspondence: Paul A. Egan

<EMAIL_ADDRESS>
#### †Present address:

Evan C. Palmer-Young, Department of Entomology, University of California, Riverside, Riverside, CA, United States

#### Specialty section:

This article was submitted to Functional Plant Ecology, a section of the journal Frontiers in Plant Science

Received: 27 April 2018 Accepted: 28 August 2018 Published: 26 September 2018

#### Citation:

Egan PA, Adler LS, Irwin RE, Farrell IW, Palmer-Young EC and Stevenson PC (2018) Crop Domestication Alters Floral Reward Chemistry With Potential Consequences for Pollinator Health. Front. Plant Sci. 9:1357. doi: 10.3389/fpls.2018.01357 Crop domestication can lead to weakened expression of plant defences, with repercussions for herbivore and pathogen susceptibility. However, little is known about how domestication alters traits that mediate other important ecological interactions in crops, such as pollination. Secondary metabolites, which underpin many defence responses in plants, also occur widely in nectar and pollen and influence plantpollinator interactions. Thus, domestication may also affect secondary compounds in floral rewards, with potential consequences for pollinators. To test this hypothesis, we chemically analysed nectar and pollen from wild and cultivated plants of highbush blueberry (Vaccinium corymbosum L.), before conducting an artificial diet bioassay to examine pollinator-pathogen interactions. Our results indicated that domestication has significantly altered the chemical composition of V. corymbosum nectar and pollen, and reduced pollen chemical diversity in cultivated plants. Of 20 plant metabolites identified in floral rewards, 13 differed significantly between wild and cultivated plants, with a majority showing positive associations with wild compared to cultivated plants. These included the amino acid phenylalanine (4.5 times higher in wild nectar, 11 times higher in wild pollen), a known bee phagostimulant and essential nutrient; and the antimicrobial caffeic acid ester 4-O-caffeoylshikimic acid (two times higher in wild nectar). We assessed the possible biological relevance of variation in caffeic acid esters in bioassays, using the commercially available 3-O-caffeoylquinic acid. This compound reduced Bombus impatiens infection by a prominent gut pathogen (Crithidia) at concentrations that occurred in wild but not cultivated plants, suggesting that domestication may influence floral traits with consequences for bee health. Appreciable levels of genetic variation and heritability were found for most floral reward chemical traits, indicating good potential for selective breeding. Our study provides the first assessment of plant domestication effects on floral reward chemistry and its potential repercussions for pollinator health. Given the central importance of pollinators for agriculture, we discuss the need to extend such investigations to pollinator-dependent crops more generally and elaborate on future research directions to ascertain wider trends, consequences for pollinators, mechanisms, and breeding solutions.

Keywords: domestication, floral rewards, Vaccinium, crop evolution, pollinator-pathogen interactions, Bombus impatiens, pollinator health, phytochemicals

## INTRODUCTION

fpls-09-01357 September 25, 2018 Time: 17:25 # 2

Humans have domesticated and selectively bred crops for millennia (Smith, 2001). While domestication has delivered many agronomic benefits, including enhanced yield and other desirable traits (Evans, 1996; Bai and Lindhout, 2007; Dempewolf et al., 2017), negative or unintended consequences of this process have included reduced genetic diversity (Van de Wouw et al., 2010; Meyer et al., 2012), and the impaired function or disappearance of potentially useful traits (Rosenthal and Dirzo, 1997; Li et al., 2017) such as physical or chemical defences against herbivory and disease (Wink, 1988; Jones, 1998; Gols et al., 2008; Sujana et al., 2012). Plant defence traits can impose considerable resource allocation costs, and can thereby trade-off with fitness or yield (Kempel et al., 2011). Hence, where selection is consistently imposed over many generations to maximise crop yield, agronomic gains may accrue at the risk of compromising other traits from which resources are diverted (Rosenthal and Dirzo, 1997; Brown and Rant, 2013). It is only relatively recently, however, that the extent of domestication impacts on crop ecological function has come to be fully appreciated (Meyer et al., 2012; Chen et al., 2015; Whitehead et al., 2017). This topic has garnered increased attention due to growing interest in the agronomic potential of traits traditionally neglected in crops, such as herbivore-induced plant volatiles (HIPVs) and extrafloral nectaries involved in the attraction of pest natural enemies (Rasmann et al., 2005; Rodriguez-Saona et al., 2009; Chen et al., 2015; Heil, 2015; Li et al., 2017).

In contrast to impacts on crop interactions with herbivores and pathogens, very little is known about the effect of domestication on interactions with pollinators and the traits which mediate these interactions. What little is known on this topic to date has mostly come from the study of ornamental plants, where widespread loss of floral scent is a reported consequence of breeding (Vainstein et al., 2001; Pichersky and Dudareva, 2007). This lack of information on domestication effects on pollinator-relevant traits, including floral reward chemistry, is surprising, given the importance of insect pollination for agriculture. About 75% of the world's staple food crops rely to some extent on the ecosystem service of pollination (Klein et al., 2007), which contributes an estimated \$351 billion (USD) annually to global food production (Lautenbach et al., 2012). Pollinators also have the potential to contribute to human nutritional health and need for micronutrients (Chaplin-Kramer et al., 2014; Ellis et al., 2015), and improve global food security (Bailes et al., 2015).

Although historically studied separately, plant-herbivore and plant-pollinator interactions are interconnected (Strauss and Armbruster, 1997; Adler et al., 2001; Irwin et al., 2003; Strauss and Irwin, 2004; Kessler and Halitschke, 2009; Lucas-Barbosa, 2016; Muola et al., 2017; Nepi et al., 2018). For example, a broad range of defence-related secondary metabolites (including alkaloids, phenolics, and cyanogenic glycosides) are expressed in floral rewards, including nectar and pollen (Baker, 1977; Rhoades and Bergdahl, 1981; Adler, 2000; Adler and Irwin, 2005; Heil, 2011; Arnold et al., 2014; Irwin et al., 2014; Stevenson et al., 2017). These compounds range widely in concentration – from trace amounts, to concentrations similar to other plant tissues (Stevenson et al., 2017, and references therein) – and in ecological function. The ecological effects of secondary compounds in floral rewards include warding off nectar robbers and microbial growth (Irwin et al., 2004; González-Teuber and Heil, 2009; Barlow et al., 2017), enhancing pollinator memory (Wright et al., 2013), and reducing pollinator infection by pathogens and parasites (Baracchi et al., 2015; Richardson et al., 2015; Erler and Moritz, 2016; Palmer-Young et al., 2016). Evidence now suggests that secondary metabolites occur ubiquitously in floral rewards, and commonly show phenotypic integration across herbivore- and pollinator-consumed materials in wild and cultivated plants (Adler et al., 2006, 2012; Kessler and Halitschke, 2009; Manson et al., 2012; Cook et al., 2013; Richardson et al., 2016; Palmer-Young et al., in press). It is therefore of great interest to assess whether domestication effects on plant defence are also mirrored for floral reward chemistry, with potential ramifications for ecological function.

We used blueberry as a crop system to examine the effect of domestication on floral reward chemistry and its potential consequence for pollinators. We included in this study wild and cultivated plants of Vaccinium corymbosum L. (highbush blueberry; Ericaceae), including cultivated hybrid and introgressed crosses with V. angustifolium Ait. (lowbush blueberry). The relatively recent and well-documented history of domestication of Vaccinium species (including cranberry, lingonberry, and bilberry) has rendered the genus a useful model for the study of domestication effects (Rodriguez-Saona et al., 2011; Song and Hancock, 2011; Rivera et al., 2015, 2016; Hernandez-Cumplido et al., 2018). The history of domestication of highbush and lowbush blueberry dates back to 1908 and 1909, respectively, when the first wild plants were collected from Greenfield, New Hampshire, United States, for cultivation and selective breeding (Gough, 1993). This initial highbush collection, together with two additional wild selections from New Jersey (Ehlenfeldt, 2009), formed some of the first early cultivars of V. corymbosum ('Brooks,' 'Sooy,' 'Rubel'). Together with their crosses ('Katherine,' 'Pioneer'), these first selections continue to make the largest genetic contributions to modern highbush cultivars (Ehlenfeldt, 1994; Lobos and Hancock, 2015), which form the basis of the global blueberry industry.

We examined nectar and pollen chemistry in eight cultivars or interspecific crosses of V. corymbosum, and as a basis for comparison, sampled from three wild populations within ca. 40 km of where the original founding cultivar 'Brooks' was collected. To examine potential domestication effects on pollinator health via changes in the pathogens of pollinators, we conducted an artificial diet bioassay with the common eastern bumble bee, Bombus impatiens Cresson. Bees were infected with the pathogen Crithidia, before manipulating dietary levels of a caffeic acid ester at concentrations representative of wild and cultivated plant nectar to compare their effect on infection. Crithidia species are Trypanosomatid gut pathogens that infect Bombus species via faecal-oral contamination (Durrer and Schmid-Hempel, 1994). Infection by Crithidia can reduce learning (Gegear et al., 2005, 2006), colony growth rates (Shykoff and Schmid-Hempel, 1991) and queen founding

success (Brown et al., 2003), and is associated with reduced reproduction in wild bumble bee colonies (Goulson et al., 2018). In Massachusetts, United States Crithidia can infect up to 80% of Bombus (Gillespie, 2010).

Specifically, we tested the following hypotheses: (1) Domestication has altered chemical composition and reduced chemical diversity of nectar and pollen; (2) Nectar and pollen chemical traits show genetic variation and heritability, but cultivars are phenotypically less variable than wild populations, consistent with their clonal propagation; (3) Nectar and pollen chemical concentration and composition are genetically correlated, which could constrain targeted breeding for floral reward chemistry; and (4) Altered expression of nectar antimicrobial compounds, such as caffeic acid esters, can influence pollinator-pathogen interactions.

### MATERIALS AND METHODS

#### Plant Material and Field Sampling

Eight cultivars were selected for inclusion in this study, based on suitable replication across sampled farms: 'Patriot' (P), 'Reka' (R), 'Liberty' (L), 'Bonus' (B), 'Friendship' (F), 'Northland' (N), 'BlueCrop' (BC), and 'Spartan' (S). The genetic origin of the majority of these cultivars is pure V. corymbosum (cultivars B, L, N, R, and S), whereas cultivars BC, F, P are interspecific hybrids with minor parentage from V. angustifolium (**Supplementary Table 1**). A range of ploidy levels are known to exist within the genus Vaccinium. However, commercially grown highbush cultivars, including those with minor percentages of V. angustifolium, are reported to be tetraploids (Rowland and Hammerschlag, 2005). The cultivars included in this study, and domesticated highbush plants generally, are typically propagated by vegetative means (softwood or hardwood cuttings) (Gough, 1993), meaning that all sampled plant individuals of the same cultivar were genetic clones.

Nectar and pollen from wild and cultivated highbush blueberry were sampled in western Massachusetts, United States, in May 2014. Wild samples were collected from three sites at least 5 km apart between May 15 to 26 [Harvard Forest (HF): 42◦ 320 6 <sup>00</sup>N, 72◦ 110 1900W, Harvard Pond (HP): 42◦ 290 5400N, 72◦ 120 4700W, and Quabbin Reservoir (Q): 42◦ 230 3200N, 72◦ 240 1100W]. We sampled cultivars at several farms between May 19 and 28 from Cold Spring Orchard (42◦ 150 6 <sup>00</sup>N, 72◦ 210 3800W; cultivars P, R, L, B, F, and N), Kenburn Orchard (42◦ 360 4300N, 72◦ 390 1800W; cultivar P), Nourse Farms (42◦ 250 4900N, 72◦ 350 1800W; cultivars BC and S) and Sobieski's River Valley Farm (42◦ 270 1200N, 72◦ 350 4300W; cultivars BC and S). To ensure no large confounding effect of sampling across multiple farms, cultivar-by-farm interactions were tested but were not significant (data not shown) for nectar and pollen chemical composition and diversity, as quantified below. For analyses other than nectar and pollen chemical composition and diversity, we included only cultivars which had been sampled from a single farm (Cold Spring Orchard).

Ten wild plants were sampled per site, and 5 cultivated plants per cultivar per farm. To prevent pollinator access to nectar, flowers were bagged with mesh usually one day before collection for all plants except at Sobieski's farm, where we obtained sufficient nectar without bagging; sometimes bags were left on plants for several days if weather was not conducive to collection (e.g., rain). Where a mixture of bagged and unbagged samples were collected for a cultivar (i.e., BC and S), we tested to affirm that bagging did not hold a significant effect on compound concentrations (data not shown). Nectar was typically collected from flowers using 2 µl glass microcapillary tubes inserted into the flower. Nectar was pooled across flowers within plants with a target of 20 µl per plant (range: 6.76–32.16 µl), and added to 80 µl ethanol in a 1.5 ml microcentrifuge tube. Anthers were collected using forceps, and thus our samples included the pollen sac and a small amount of filament in addition to granular pollen. We collected at least 5 mg per plant by pooling across flowers within plants. Nectar and pollen samples were placed on ice in a cooler in the field, frozen at −20◦C, and then freeze-dried prior to analysis.

### Chemical Analysis

Freeze-dried nectar was redissolved in 50 µL MeOH. For extraction of pollen, dried samples were sonicated for 10 min in 1 ml MeOH, and incubated at room temperature for 24 h. Samples were then centrifuged at 13,000 rpm for 5 min, and supernatant transferred to glass vials for analysis. Sample analyses were performed by liquid chromatography electrospray ionisation mass spectroscopy (LC-ESIMS) and UV spectroscopy using a Micromass ZQ LC-MS (Waters, Elstree, Herts, United Kingdom). Aliquots of nectar were injected directly onto a Phenomenex (Macclesfield, Cheshire, United Kingdom) Luna C18(2) columns (150 × 3.0 mm i.d., 5 um particle size) and compounds eluted using MeOH (A), H2O (B) and formic Acid (C) with A = 0%, B = 90% at T = 0 min; A = 90%, B = 0% at T = 20 min and held for 10 min with C at 10% throughout the analyses. Column temperature was 30◦C with flow rate = 0.5 ml min−<sup>1</sup> . High resolution MS spectra were used to provide additional data for compound identification and were recorded for a subset of samples using a Thermo LTQ-Orbitrap XL mass spectrometer (Waltham, MA, United States) with compound separation on an Accela LC system using similar elution parameters as described above. Except for identification of caffeoylquininc and caffeoylshikimic acids (see below), compounds were identified by comparison with mass spectra available via the NIST spectral database version 2.0 and with authentic standards available at Royal Botanic Gardens, Kew. Compounds were quantified using calibration curves with authentic standards based on UV absorbance or compound ionisation in the mass spectrometer. Where these were not available, compound quantities were determined using standard curves calculated from the UV absorbance of compounds with the same chromphore.

These methods also permitted the detection of two amino acids, phenylalanine and tryptophan. For convenience, we used the term 'total amino acids' to refer to the two amino acids only detected and quantified. Compound concentrations are for nectar expressed in µmol L−<sup>1</sup> original volume, and for pollen in

µmol kg−<sup>1</sup> dry mass. We refer to 3-, 4-, and 5-O-caffeoylquinic acids (CQAs) collectively as chlorogenic acids, and specifically to 3-CQA as chlorogenic acid.

Our LC-MS analysis identified CQAs as principal secondary metabolites in nectar based on their mass spectral properties, which were similar to those reported previously for these compounds (Schütz et al., 2004). This identification was supported by comparison with authentic standards from our own in-house (Jodrell Laboratory) collection, and their accurate mass pseudomolecular ion of m/z 355.1038 which gave a protonated molecular formula [M+H]<sup>+</sup> of C16H19O9. CQAs are esters of caffeic acid with a hydroxylated cyclohexanoic acid, quinic acid (Schütz et al., 2004). Our analysis also identified an additional caffeic acid ester in nectar of V. corymbosum which had a similar UV spectrum to the CQAs (indicative of a caffeoyl moiety). However, this ester recorded a pseudomolecular ion of m/z 337.0926 (calcd. 337.0918), corresponding to a protonated molecular formula [M+H]<sup>+</sup> of C16H17O8, suggesting a dehydro derivative of CQA. Further comparison of the (-) MS2 data showed major fragment ions at m/z 291 and 179, indicative of caffeoylshikimic acids as reported in Phoenix dactylifera (Habib et al., 2014). Subsequent co-elution with an authentic laboratory standard and (-) MS2 showing similar fragments at 179, 135, 161, and 291 allowed assignment of the compound to 4-Ocaffeoylshikimic acid (4-CSA).

#### Bumblebee Infection Assay

As per our chemical analysis, 4-CSA largely differentiated wild and cultivated plants of V. corymbosum (see section "Results"), and thus warranted further biological evaluation. We tested chlorogenic acid (3-CQA) as a proxy for this compound, since no commercial source of 4-CSA was available, and different caffeic acid esters typically show equivalent biological activities (Guzman, 2014), likely owing to their common ortho-dihydroxy substitution (Stevenson et al., 1993). For example, in bioassays against various species of bacteria and yeast, 3-CQA, 5-CQA, and other caffeic acid esters frequently possessed identical minimum inhibitory concentration (MIC) values (Zhu et al., 2004; Xia et al., 2011; Guzman, 2014).

Bioassays were conducted with workers of Bombus impatiens parasitized with Crithidia. To test how 3-CQA affected Crithidia infection, we experimentally infected Bombus impatiens workers before feeding them 30% sucrose solution without or with 3- CQA (C16H18O9; molecular weight 354.31 g/mol; CAS number 327-97-9; Sigma-Aldrich, St. Louis, MO, United States) at two concentrations, low (2.8 µM [1 ppm] 3-CQA dissolved in 30% sucrose solution), and high (56.5 µM [20 ppm] dissolved in 30% sucrose solution). These represented low and high concentrations of 4-CSA found naturally in cultivated (4.44 µM ± 7.52 SD) and wild (22.03 µM ± 27.46 SD) plants, respectively (see section "Results").

Thirty worker bees were tested per treatment (n = 90 bees total), evenly spread across three source colonies (Biobest Canada, Leamington, Canada; 10 bees per source colony per treatment). Bees were removed from their source colonies, randomly assigned to treatments, starved for 4–6 h, and then experimentally infected with Crithidia on the same day. Crithidia were isolated from wild B. impatiens collected in Massachusetts, United States (GPS coordinates: 42◦ 240 2500N, 72◦ 310 4600W), and maintained in B. impatiens colonies in the laboratory. Crithidia inoculum was prepared according to a standard protocol (Richardson et al., 2015). In brief, on the day of experimental inoculation, 10 workers were sacrificed from the infected colonies and their intestinal tracts ground individually in 300 µl of dH20. After vortexing the samples, we allowed them to settle at room temperature for 4 h. We then placed 10 µl of the clear solution on a haemocytometer and examined it for Crithidia. For samples that contained Crithidia, we transferred 50–150 µl of solution to a clean container and diluted to make an approx. 30% sucrose solution with 6000 Crithidia cells × 10 µl −1 . Each bee was provisioned with 10 µl of inoculum containing 6000 Crithidia cells, which is within the range of concentrations of the pathogen that bees encounter (e.g., Schmid-Hempel and Schmid-Hempel, 1993; Otterstatter and Thomson, 2006). Immediately following inoculation, bees were housed individually in plastic containers. They were given one lump of honey bee collected pollen (Koppert Biological Systems, Howell, MI, United States) mixed with sugar water, and new sugar solution ad libitum of the appropriate treatment. Pollen and nectar were replaced daily for 7 days.

After 7 days, we sacrificed individuals and prepared intestinal tracts as when making Crithidia inoculum. We placed 10 µl of intestinal solution on a haemocytometer and counted the number of Crithidia cells in five subfields of the grid at 10x magnification, totalling 0.02 µl, with a dissecting microscope. We summed these counts and calculated the number of Crithidia cells per mL for each bee. We also removed the right forewing of each bee, mounted it on a microscope slide, and measured the radial cell length as an estimate of bee size (Harder, 1982).

## Statistical Analyses

#### Chemical Composition

Comparisons of chemical composition between wild and cultivated plant nectar and pollen were made on both an absolute and proportional basis, which describe two different aspects of composition. Hence, the quantitative concentration of compounds was used directly in the absolute composition analysis, whereas the relative concentration of compounds (their percent contribution on a molar basis relative to other compounds in the sample) was used in proportional composition analysis. For this, a proportional dataset was generated in which compound concentrations within a sample row were standardised by dividing each by the row total. Absolute composition was examined by plotting principle components analysis (PCA) ordinations using the 'rda' function in the R package 'vegan' (Oksanen et al., 2017). PCA provided good representation of the multivariate composition of nectar and pollen, in which the cumulative proportion of variance explained by the first two components was 0.84 and 0.65, respectively. For proportional analyses, unconstrained NMDS (Non-metric multidimensional scaling) ordinations were fitted and plotted based on a Bray-Curtis distance metric in 'vegan.'

As a complement to ordinations, the hypothesis that wild and cultivated plants differed in nectar and pollen chemical composition was explicitly tested using permutational multivariate ANOVAs (function 'adonis' in vegan). For this, distance matrices were first generated for absolute composition based on Euclidean distance (to better preserve quantitative relationships), and for proportional composition using Bray– Curtis distance. Similar to univariate ANOVA, this technique relies on the assumption of multivariate homogeneity between groups (i.e., between wild and cultivated plants). According to homogeneity of multivariate dispersions tests (vegan function 'betadisper'), this assumption was met for all variables except nectar proportional composition. As an alternative to permutational multivariate ANOVA, we therefore opted to compare nectar proportional composition based on axis scores extracted from NMDS ordination. NMDS axis 1 and 2 scores provided a reduced dimensionality representation of proportional composition, and were each fitted as a univariate response in generalised linear mixed models (GLMMs) using the R package 'nlme' (Pinheiro et al., 2017). The GLMMs were fitted with an explanatory variable of 'origin' (wild or cultivated), and a nested random effect of group ID (cultivar or population name). A variance structure was added to both models via the 'weights' function of nlme, to control for unequal variances between wild and cultivated plants. Models were otherwise validated by examining standardised residuals for normal distribution.

'Indicator compound analysis' (or multilevel pattern analysis) was implemented using R package 'indicspecies' (Cáceres and Legendre, 2009) to further probe any potential differences in chemical composition between wild and cultivated plants. This analysis used a permutational approach to test for compound association (or differences in abundance) between different groups (De Cáceres, 2013), in our case wild and cultivated plants. Compound association values (i.e., point-biserial correlation coefficients) and their corresponding p-values (testing the null hypothesis that the correlation is zero) were generated using the 'multipatt' function, based on 999 permutations, and use of a corrected correlation coefficient (specified as 'r.g' in multipatt). This correlation coefficient is similar to Pearson correlation, and is likewise bounded between zero and one.

#### Chemical Diversity