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- Materials and methods
Plants face varying pressures from herbivores as they progress through their life cycle. A large body of work has examined the ontogeny of plant defence and the ways in which it is shaped by stage- or size-specific selection by herbivores (reviewed in Boege & Marquis 2005; Barton & Koricheva 2010). Examining ontogenetic variation in defence can inform understanding of how plants resolve optimality problems associated with allocation to growth and defence over the life cycle. Much of this work has focused on physical or chemical defences (e.g. Ohnmeiss & Baldwin 2000). In addition, ontogenetic variation in indirect (biotic) defences, particularly traits that mediate defensive mutualisms with ants, has received increasing attention (Heil et al. 2000; Trager & Bruna 2006; Pringle, Dirzo & Gordon 2012; Villamil, Marquez-Guzman & Boege 2013). Just as ontogenetic variation in direct defence is important for understanding plant–herbivore interactions (Barton & Koricheva 2010), variation in indirect defence traits may be important for understanding the dynamics of plant–animal mutualisms.
Extrafloral nectar (EFN) is a common currency with which ant protection is traded. This indirect defence strategy is employed widely across plant families (Koptur 1992; Marazzi, Bronstein & Koptur 2013). EFN is a complex reward that can vary in many dimensions, including rate of secretion, concentrations of total carbohydrates (CHs) and free amino acids (AAs), relative abundances of constituent CHs and AAs, and even secondary compounds and active enzymes (Heil 2011). Identifying sources of variation in these EFN traits is important because they can influence the quantity and quality of ant defence. For example, ant patrolling tends to increase with rate of EFN secretion (Kost & Heil 2005; Villamil, Marquez-Guzman & Boege 2013), and partner identity and their aggression towards herbivores can vary with CH and AA composition (Blüthgen & Fiedler 2004; Gonzalez-Teuber & Heil 2009; Ness, Morris & Bronstein 2009; Wilder & Eubanks 2010; Shenoy et al. 2012). The relative abundance of monosaccharide vs. disaccharide sugars has emerged as a particularly important dimension of EFN variability because ant species can differ in their abilities to digest disaccharides (Heil, Rattke & Boland 2005; Kautz et al. 2009). Any EFN traits that influence ant species identity could have consequences for defence because ant species are often unequal in their abilities to protect plants from herbivores (e.g. Ness, Morris & Bronstein 2006).
While EFN traits can clearly have important ecological consequences, we are just beginning to understand how these traits vary with plant demographic state (size or life stage) and translate to variation in realized defence. Studies across a variety of systems have shown that EFN production (or ant activity, often used as a proxy for EFN production) increases with plant size or age and is greater on reproductive plants or plant parts than on vegetative plants or plant parts (Wäckers & Bonifay 2004; Trager & Bruna 2006; Miller 2007; Shenoy et al. 2012; Villamil, Marquez-Guzman & Boege 2013). Recent studies also suggest that CH and AA concentration and composition can change with reproductive state, with generally sweeter nectar on reproductive vs. vegetative plants or plant parts (Shenoy et al. 2012; Villamil, Marquez-Guzman & Boege 2013). Associations between EFN quantity/quality and plant size and reproductive state have been interpreted in the light of optimal defence theory (e.g. Wäckers & Bonifay 2004; Holland, Chamberlain & Horn 2009), which predicts that plants should maximize defence of structures that contribute most to fitness (Rhoades 1979).
Shifts in EFN traits associated with plant reproduction may be manifested at multiple scales. At a smaller scale, there may be differences in the amount or type of EFN secreted by individual EFNs on reproductive structures vs. nectaries on vegetative plant parts (e.g. Wäckers & Bonifay 2004; Shenoy et al. 2012). At a larger scale, the onset of reproduction may lead to different amounts or types of EFN secreted from vegetative vs. reproductive plants (Villamil, Marquez-Guzman & Boege 2013). Finally, the ways in which nectary-level traits scale up to influence rewards at the whole-plant level depend on how the total number of nectaries and relative allocation between vegetative and reproductive structures scale with plant development. No previous studies have integrated the influence of plant size and reproductive status over multiple scales. Furthermore, because reproductive status is often positively correlated with plant size, the relative contributions of these variables to ontogenetic variation remain unclear.
Unlike direct defences, EFN is both a plant defence trait and a resource that mediates multispecies interactions (Rudgers & Gardener 2004; Holland, Chamberlain & Miller 2011) and is therefore important to consider from the ants' perspectives as well as the plants' (Lanan & Bronstein 2013). Plant size- or stage-related variation in EFN production and composition may have consequences for the guild of ant partner species that rely on this reward. For example, ontogenetic shifts in ant partner identity have emerged as an intriguing pattern across ant–plant defensive mutualisms, with different ant species associating non-randomly with different plant life stages (Young, Stubblefield & Isbell 1997; Fonseca & Benson 2003; Djieto-Lordon et al. 2004; Miller 2007; Palmer et al. 2010; Miller & Rudolf 2011). Little is known about mechanisms underlying these patterns. Whether variation in plant rewards underlies shifts in partner association remains an open question. At the population level, plant size- or stage-related variation in EFN could generate resource heterogeneity, potentially expanding opportunities for coexistence of multiple ant partners (Young, Stubblefield & Isbell 1997; Lee & Inouye 2010). Genotype differences could also contribute to population-level variation in rewards (e.g. Ballhorn, Godschalx & Kautz 2013).
In this study, I quantified variation in EFN quantity and quality in a long-lived desert plant, the tree cholla cactus, Opuntia imbricata Haw. [D.C.]. While ‘ontogenetic variation’ is widely used in the plant defence literature, here I focus on plant size and reproductive state (vegetative or flowering), two important axes of ontogeny. I examined multiple dimensions of EFN variability, including presence/absence of EFN, rate of secretion, concentrations of total CHs and AAs, and relative abundances of component CHs and AAs across plant sizes and stages. I employed a sampling design and statistical approach that allowed me to quantify the independent and interactive effects of plant size and reproductive state and test for their effects at small (nectary-level) and large (plant-level) scales. To connect nectar traits to ant–plant interactions, I also documented associations between plant demographic state and interactions with ants, including visitation and species identity.
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- Materials and methods
Previous studies have documented variation in EFN traits associated with various measures of ontogeny: leaf age (Heil et al. 2000; Miller, Legaspi & Legaspi 2010); plant size or age (e.g. Trager & Bruna 2006); vegetative vs. reproductive plant stages (e.g. Villamil, Marquez-Guzman & Boege 2013); and vegetative vs. reproductive organs within reproductive plants (e.g. Wäckers & Bonifay 2004; Shenoy et al. 2012). To my knowledge, this is the first study to integrate multiple components of EFN quantity and composition over multiple dimensions of ontogenetic variation and connect them to patterns of ant–plant interactions in the field. I found that EFN traits at the level of individual nectaries varied significantly with plant size and nectary type. Furthermore, nectary-level variation scaled up to affect rewards at the whole-plant level, such that the total quantity and quality of EFN provided to ant partners depended on a plant's demographic state (size, reproductive status and their interaction). Finally, there were significant associations between plant demographic state and ant visitation, suggesting that demographic variation in EFN rewards can modify ant–plant interactions. Demographic structure is central to most studies of plant population dynamics but is rarely explicitly considered in the context of plant–animal mutualisms (Miller & Rudolf 2011). The results of this study identify plant demographic structure as an important source of variation in rewards, with implications for plant defence and the ecological dynamics of multispecies mutualisms.
In general, variation in EFN quantity was explained by a combination of plant size and reproductive state, whereas variation in EFN composition was dominated by reproductive state alone. However, there were complex details underlying these general trends stemming from interactions between size and state, the traits affected, and the scales at which effects were manifest. At the scale of individual nectaries, the probability of producing any EFN showed the greatest variation with respect to size and was additionally greater for reproductive vs. vegetative nectaries. By contrast, the rate of secretion for nectaries with nonzero EFN differed between nectaries on vegetative vs. reproductive structures but not with respect to size. Nectary type also dominated variation in EFN CHs and AAs, with weaker effects of size. The influence of reproduction was manifest in the contrast between vegetative vs. reproductive nectaries within individuals and/or between the vegetative nectaries of vegetative vs. reproductive plants, depending on the response variable. Most of these nectary-level traits scaled up to influence various dimensions of partner quality at the whole-plant level. Thus, plant rewards clearly varied with ontogeny, but in different ways for different reward traits, at different scales and for different axes of ‘ontogeny’. These results provide a nuanced perspective on how plant investment in biotic defence varies among plant structures and over the course of the life cycle.
My results are consistent with prior studies demonstrating increasing investment in traits associated with defensive mutualism as plants develop (Fiala et al. 1994; Trager & Bruna 2006; Kwok & Laird 2012; Villamil, Marquez-Guzman & Boege 2013), raising questions about the ultimate causes of ontogenetic patterns. In some cases, resource limitation and/or architectural constraints may limit investment by young plants in rewards for defensive mutualists. For example, myrmecophytic plants may be unable to support symbiotic ant defenders until reaching a minimum size for production of domatia (Fiala et al. 1994). Villamil, Marquez-Guzman & Boege (2013) found that structural characteristics made the EFNs of juvenile Turnera velutina physiologically incapable of secreting EFN. However, it is also important to consider selection by herbivores and the adaptive value of defences that are specific to plant stages or structures (i.e. optimal defence theory: Rhoades 1979; Wäckers & Bonifay 2004; Holland, Chamberlain & Horn 2009). In this system, insect herbivory is infrequent on small tree cholla, but herbivore pressure increases significantly with size/age and especially with the onset of reproduction (Miller 2007). The elevated risk of herbivory for mature plants may select for lower investment in defensive mutualism when small, given that EFN may be costly to produce (Rutter & Rausher 2004) but yield little fitness benefit.
In addition to size-dependent EFN production, there were strong signatures of reproduction, independent of size, on EFN quantity and quality from the nectary to whole-plant scales. Cacti at reproductive life stages produce large amounts of floral nectar to attract and reward insect pollinators (Scogin 1985; McFarland, Kevan & Lane 1989). Strong correlation between the production of floral and extrafloral nectar is probably not coincidental. Chamberlain & Rudgers (2012) found positive correlations between floral and EFN traits across species in the genus Gossypium. They concluded that the two sets of traits covary due to common genetic and/or physiological bases. Indeed, floral and extrafloral nectaries are not physiologically different, and they share regulatory cues, including jasmonic acid (JA) (Heil 2011). Mechanisms that account for positive correlations across species may therefore also explain positive correlations across developmental stages within species. In addition to the possibility of being a ‘side-effect’ of flowering, I hypothesize an adaptive value to the shift in EFN traits associated with reproduction. Increased EFN secretion can increase ant patrolling, and this could equip reproductive plants (which face elevated pressure from floral-feeding insects) with enhanced defence, purely in terms of ant numbers.
Additionally, field surveys showed that reproductive plants differed not only in ant numbers but also in ant identity, with reproductive plants being tended almost exclusively by L. apiculatum. This could further enhance defence of reproductive plants because L. apiculatum is a superior bodyguard to C. opuntiae (Miller 2007). Furthermore, ant tending deters tree cholla pollinators and C. opuntiae imposes a stronger pollination cost than does L. apiculatum (Ohm and Miller, unpubl. manuscript). Thus, not only is C. opuntiae a poor defender, but it may even have a net parasitic effect at reproductive life stages. Are tree cholla simply lucky to associate non-randomly with L. apiculatum at the life stages when this partner is most beneficial and least costly? Or are there adaptive mechanisms by which plants can ‘choose’ partners?
Based on the data, I suggest two possible mechanisms for an influence of EFN traits on partner identity. First, the significant increases in EFN quantity and quality associated with reproduction may allow plants to exploit the ant competitive hierarchy. Large, reproductive individuals are the most valuable plant partners but these are rare in the population and their high rate of ant occupancy (100%) suggests that they are a limiting resource for which ants compete strongly. Liometopum apiculatum is not only a superior defender to C. opuntiae but also a superior competitor (Miller 2007). The high prevalence of L. apiculatum on reproductive plants could represent competitive exclusion of C. opuntiae. Thus, positive correlation between competitive and defensive abilities could mean that when ants compete, plants win. Secondly, the change in disaccharide content of reproductive plants may influence partner identity based on ant production of invertase, the enzyme that cleaves disaccharide sugars into monosaccharides (necessary for digestion). Co-variation across plant and ant species between EFN disaccharide content and ant invertase activity can explain partner-specific associations in ant-Acacia mutualisms (Heil, Rattke & Boland 2005; Kautz et al. 2009). Whether a similar mechanism can explain life stage-specific associations in this system remains an open question. These hypotheses merit experimental investigation, particularly given our poor understanding of shifts in partner identity across plant life stages in other ant–plant mutualisms (Young, Stubblefield & Isbell 1997; Fonseca & Benson 2003; Dejean et al. 2008). Ontogenetic partner shifts have important implications for the net influence of the ant partner guild on plant fitness (Palmer et al. 2010) and for the maintenance of partner species diversity based on feedbacks between the partner guild and plant population stage structure (Lee, Miller & Inouye 2011).
Like any observational study, alternative interpretations for the patterns I documented warrant consideration. For example, differences in EFN traits between vegetative and reproductive plants may be due not (or not entirely) to reproduction per se but to unmeasured factors (e.g. microhabitat characteristics) that independently affect plant reproduction and EFN (although, importantly, within-plant contrasts control for such effects). Similarly, associations between plant demographic state, EFN traits and ant occupant could be explained by multiple pathways of causation. In addition to the influence of EFN traits on ant identity suggested above, partner identity could affect the demographic state of the plant (Vasconcelos & Davidson 2000), and unmeasured factors could independently affect ant identity and plant demographic state. Experimental approaches that disentangle these non-mutually exclusive hypotheses would be valuable. Furthermore, I do not know whether EFN is inducible in this system, as in other EFN-secreting cacti (Holland, Chamberlain & Horn 2009). It is possible that past herbivory contributed to differences between vegetative and reproductive plants, because these tend to carry different herbivore loads.
In summary, my results suggest that this and likely other plant–animal mutualisms are best viewed through the lens of demography. Populations of long-lived plants are demographically heterogeneous – spanning sizes and reproductive states – and the rewards offered to animal mutualists can track demographic heterogeneity. The ontogenetic trajectories of individual plants include significant variation in reward quantity and quality – arising from nectary-level traits that can scale up to affect whole-plant rewards – with consequences for the strength of biotic defence over the life cycle. From the perspectives of the two ant partners, the uneven distribution of demographic states within the plant population makes for a heterogeneous resource base that could affect their individual population dynamics as well as their competitive dynamics. Through their effects on herbivores and hence plant demography, ants may also influence the distribution of demographic states, setting the stage for potentially complex feedbacks that warrant attention from empiricist and theorists.