ID number: XXXXX Authcate: xxxxx Assignment 1d: Scientific Literature Review

Chemical communication resulting in defensive behaviour and seed dispersal in an obligate plant-ant mutualism
Abstract Myrmecophytes, plants that are involved in mutual relationships with ants, provide shelter and food in return for protection against herbivores, fungal pathogens, competitors and invading vines. The mermecophyte releases volatile organic compounds in order to communicate the presence of herbivores and other enemies to their ant partners. The mutualistic ants respond to these chemicals by exiting the domatia to protect the most important and vulnerable parts of the mermecophyte in turn decreasing the occurrence of herbivory. The removal of ants from their host plant shows an increase in hemipteran, herbivore and predator abundance, and a decrease in plant herbivory, reproduction and plant biomass, reinforcing the importance of these plant-ant mutualisms. Chemical cues have also been shown to induce seed carrying behaviour, also known as Mermecochory, resulting in the formation of ants-gardens. These ant-gardens are structurally reinforced by plant roots, providing a home for the ant. The plant benefits from the nutrient rich soil produced by the ant-gardens. Chemical communication between ants and plants results in an obligate mutualistic relationship by which the ant benefits from a nesting space and the plant benefits from constant protection and seed dispersal.

Keywords: Myrmecophytes, domatia, mutualistic, Mermecochory, volatile organic compounds, ant-garden

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ID number: XXXXX Authcate: xxxxx Assignment 1d: Scientific Literature Review Introduction Plants that provide food (such as extrafloral nectar) and shelter (in specialised structures called domatia) to ants are termed Myrmecophytes (Vittecoq et al. 2011). In return, the ant provides its host with protection against herbivores, fungal pathogens, competitors and invading vines (Grangier et al. 2007).

In order for host plants to be protected by the ants, there must be some way to communicate the presence of an intruder. Multiple studies have been performed to identify how mermocophytes and ants communicate and have shown it is in chemical form involving the release of VOC's (Schatz 2009; Vittecoq et al. 2011). These VOC's (volatile organic compounds) are known to allure competitors of herbivores, which is an excellent example of indirect defense (Vittecoq et al. 2011; Heil et al. 2010). The level of protection given to leaves also depends on the maturity of the leaf, older leaves appear to be more durable and need less patrolling by plant-ants (Grangier et al. 2007).

The removal of ants from their host plant has effects on hemipteran, herbivore and predator abundance, plant herbivory, reproduction and plant biomass (Rosumek et al. 2009). This is evidence that the mutualistic relationship between ants and mermocophytes is an obligate one, the ants have permanent colonies in the domatia, and rely on the plant for housing. The mermecophyte continuously relies on the ant for protection, neither the ant or the plant can survive without each other (Moraes & Vasconcelos 2009).

Plants can also benefit from mutualism with ants through the process of Mermecochory, the process by which plant seeds are dispersed by ants (Youngstedt et al. 2007). In a similar way to communication, chemicals extracts from certain plants attract ants causing seed collecting behaviour (Youngstedt et al. 2007). An example of this is seen in the lowland of the Amazonian rainforest, which are home to ant-gardens. These ant-gardens are formed by the collecting and cultivating of plant seeds by their mutualistic ant partner (Youngstedt et al. 2007). This review examines plant-ant mutualisms, firstly discussing chemical communication; secondly, the role of ants in defense mechanisms and thirdly, seed dispersal resulting in ant-gardens.

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ID number: XXXXX Authcate: xxxxx Assignment 1d: Scientific Literature Review Chemical communication Many ant-plant mutualisms have shown that when damaged, the plant releases VOC's that attract mutualistic ants resulting in a decrease in herbivory (Vittecoq et al. 2011; Schatz et al. 2009; Weir et al. 2012). Research carried out by Schatz et al. (2009) resulted in the identification of some of these VOC's. Herbivore attack was simulated using a needle to pierce leaves of Leonardoxa africana africana. The reaction of its mutualistic partner, Petalomyrmex phylax, was observed (Schatz et al. 2009). The damaged leaves along with the filter paper controls were placed 3 cm from domatia entrances in host plant branches that were inhabited by ants, and a solid phase microextraction fibre was placed 1 cm from the domatia entrances (Schatz et al. 2009). Results showed a reaction by the ants to protect the damaged leaves, but not the filter paper, confirming the presence of active VOC's (Schatz et al. 2009). Extracts from the solid phase microextraction fibre were analysed and identified the VOC's as being methyl salicylate, 2E hexen-1-ol and hexanal (Schatz et al. 2009). Further experimentation involving saturation of filter paper with synthetic VOC's showed little difference in the reaction of ants between the control and hexanal (figure 1), indicating that hexanal may not be connected to communication between ants and their host (Schatz et al. 2009).

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Figure 1: Ants left the domatia quickest when exposed to methyl salicylate and a combination of the three VOC's than when exposed to the control, 2E-hexen-1-ol and hexanal. The number of ants leaving the domatia was highest when exposed to methyl salicylate and a combination of the three VOC's, closely followed by 2E-hexen-1-ol. Hexanal and the control resulted in the least amount of ants leaving the domatia (Modified from Schatz et al. 2009).

By using electroantennography and artificially damaging leaves, Vittecoq et al. (2011) experimented on the same species as Schatz et al. (2009) and an additional mutualistic relationship between Homomyrmes afer and the mermecophyte Leonardoxa africana letouzeyi. Results for the latter were the same as in the work by Schatz et al (2009), however the relationship between H. afer and L. a. letouzeyi showed the ants only responding to hexanal. This indicates the hexanal, contrary to conclusions made by

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ID number: XXXXX Authcate: xxxxx Assignment 1d: Scientific Literature Review Schatz et al. 2009, is used in communication and that the VOC's transmitted are host specific (Vittecoq et al. 2011).

Multiple ant recruitment studies that have been carried out point to cuticular compounds providing the chemical cues for host discrimination (Weir et al. 2012; Edwards et al. 2006; Jurgens et al. 2006). The reactions of colonies of Pseudomyrmes triplarinus were compared against their host plant; Triplaris americana and closely related species Triplaris poeppigcana (Weir et al. 2012). Results indicated that not only were the ants able to identify their host, but were clearly able to differentiate between the cuticular extracts of different species (Weir et al. 2012).

The evidence provided in these studies indicates that ant-plant mutualisms communicate through VOC's in order to achieve protection against herbivory, and that the particular VOC used may differ depending on the species involved (Vittecoq et al. 2011; Schatz et al. 2009; Weir et al. 2012). Additional studies have also shown that cuticular compounds play a role in the recruitment of ant colonies to their host plant, and are used to discriminate between host species (Weir et al. 2012; Edwards et al. 2006; Jurgens et al. 2006).

Rewards for the host: defense mechanisms Mounting evidence points towards to obligate nature of plant-ant mutualisms (Moraes & Vasconcelos 2009). In their meta-analysis of ant-plant defense relationships, Rosumek et al. (2009) pooled evidence from 81 studies. Results indicated that plants without their ant partner experience twice as much herbivory as those with ants. As shown in figure 2, those plants without ants also experienced a reduction in fitness including reproduction, biomass and leaf production (Rosumek et al. 2009).

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Figure 2: Pooled experimental data indicating an increase in herbivory, herbivore abundance and predator abundance, and a decrease in plant biomass, reproduction of leaves, flowers, fruit and seeds (Modified from Rosumek et al. 2009).

Short term ant exclusion experiments lasting 40 days and long term ant exclusion experiments lasting 8 months were conducted by Grangier et al. (2009), the results of which support the findings from the Rosumek et al. (2009) meta-analysis. Colonies of Allomerus decemarticulatus were removed from all areas of their mutualistic plant partner Hirtella physophora (Grangier et al. 2009). The extent of herbivory was measured at the beginning of the experiments on already formed leaves, and on the new leaves that grew during the long term experiment (Grangier et al. 2009). Results of the short term experiment showed little increase in herbivory, compared with the long term experiment where the first three growth stages showed a significant increase in herbivory as shown in figure 3 (Grangier et al. 2009). Control plants, all containing a mature active colony of ants allowed for the growth of more new leaves (Grangier et al. 2009). The number of ants patrolling the leaves decreased with maturity indicating a decrease in vulnerability of leaves with age (Grangier et al. 2009; Heil & McKey 2003).

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Stage 1

Stage 2

Stage 3

Stage 4

Figure 3: Changes in herbivory damage on leaves during stage 1 (juvenile leaves), stage 2 (expanding leaves), stage 3 (young leaves) and stage 4 (mature leaves). A large increase in herbivory damage during the absence of ants is indicated by the dotted line (modified from Grangier et al. 2009).

An additional ant exclusion experiment was carried out by Moraes and Vasconcelos (2009) involving 24 plants, half of which were rid of their obligate ant colonies. Leaves were measured for herbivory damage at the commencement of the experiment, and each month for the five months following (Moraes & Vasconcelos 2009). Herbivore damage was shown to increase upon removal of ants as displayed in figure 4 (Moraes & Vasconcelos 2009).

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Figure 4: Leaf damage due to herbivory increased significantly upon removal of obligate ant inhabitants (Modified from Moraes & Vasconcelos 2009).

The herbivory damage of plants with obligate ants, opportunistic ants and no ants were also compared (Moraes & Vasconcelos 2009). Results as shown in figure 5 indicate those plants with opportunistic ants or no ants at all suffered twice as much damage from herbivores as plants with obligate ants (Moraes & Vasconcelos 2009).

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Figure 5: Plants inhabited by obligate ants show less herbivory damage compared to those with opportunistic ants or no ants (Modified from Moraes & Vasconcelos 2009).

These results along with the work of Grangier et al. (2009) also support the findings from Rosumek et al. (2009), plants in obligate mutualistic relationships with ants are provided with consistent protection from herbivores.

Rewards for the host: seed dispersal and ant-gardens

Mermecochory, the process by which plant seeds are dispersed by ants, is another of the ways that a plant benefits from having a mutualistic relationship with ants (Youngsteadt et al. 2008; Servigne and Detrain 2010). The lowland of the Amazonian rainforest is home to ant gardens which are formed from the collection of seeds from epiphyte plants such as Peperomia macrostachya, by aboreal ants such as Camponotus femoratus (Youngsteadt et al. 2008). After the seeds have been collected, the ants incorporate them

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ID number: XXXXX Authcate: xxxxx Assignment 1d: Scientific Literature Review into a carton structure that is rich in nutrients such as nitrogen, phosphorus and potassium (Orivel and Leroy 2011). The ants continuously nurture their gardens which assists in growth and survival of their epiphyte hosts (Schmit-Neuerburg and Bluthgen 2007). The ants also provide some herbivore defense, however it is not the induced defense that is seen in mermocophytes, rather it is a form of constitutive defense (Vantaux et al. 2007). The structure of the ant garden is reinforced by the roots of the epiphytes, which enables the plant to provide the ants with a substantial barrier against the abiotic elements (Schmit-Neuerburg and Bluthgen 2007).

In research carried out by Youngsteadt et al (2008), it was indicated that these mutualistic relationships, like those involving ants and mermocophytes, are host discriminate. Seeds that ants usually ignore had extracts from Peperomia macrostachya applied, these seeds along with control seeds treated with a solvent were placed 5 cm from foraging trails of C. femoratus (Youngsteadt et al. 2008). Observations of the ants reactions showed the control did not cause seed carrying behaviour while the extracts from the plant did (Youngsteadt et al. 2008). Moreover, results showed that ants were more attracted to the odour of Peperomia macrostachya seeds than that of the control seeds, indicating that chemical cues are adequate in attracting ants and causing seed collecting behaviour (Youngsteadt et al. 2008).

The evidence provided from research concerning Mermecochory and the construction of ant gardens supports the findings that these mutual relationships are obligate and are based on chemical cues, not unlike the mutualisms between ants and mermocophytes (Youngsteadt et al. 2008; Vantaux et al. 2007; Schmit-Neuerburg and Bluthgen 2007).

Conclusion Having reviewed research surrounding obligate plant-ant mutualisms, it is evident that chemical communication is the primary way that plants and ants interact with each other in order to gain the benefits of protection and seed dispersal (Scharz et al. 2009; Vittecoq et al. 2011; Weir et al. 2012; Youngsteadt et al. 2008). The evidence provided in these studies indicates that ant-plant mutualisms undergo different effects depending on the

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ID number: XXXXX Authcate: xxxxx Assignment 1d: Scientific Literature Review types of species involved, but regardless of this, all obligate mutualisms between ants and plants see a decrease in the levels of herbivory and an increase in plant fitness (Rosumek et al. 2009; Grangier et al. 2008; Vittecoq et al. 2011).

Word count: 1938

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ID number: XXXXX Authcate: xxxxx Assignment 1d: Scientific Literature Review

References
Edwards, DP, Hassal, M & Sutherland, DWY (2006) Assembling a mutualism: ant symbionts locate their host plants by detecting volatile chemicals. Insectes Sociaux 53: 172-176. Grangier, J, Dejean, A, Male, PJG & Orivel, J (2008) Indirect defence in highly specific ant-plant mutualism. Naturwissenschaften 95: 909-916.

Heil, M & McKey , D (2003) Protective ant-plant interactions as modern systems in ecological and evolutionary research. Annual Review of Ecology, Evolution and Systematics 34: 425-453.

Heil, M, Orona-Tamayo, D, Eilmus, S, Kautz, S & Gonzalez-Teuber, G (2010) Chemical communication and co-evolution in an ant-plant mutualism. Chemeoecology 20: 6374.

Jurgens, A, Feldhaar, H, Feldmeyer, B & Fiala, B (2006) Chemical composition of leaf volatiles in Macaranga species (Euphorbiaceae) and their potential role as olfactory cues in host localization of foundress queens of specific ant partners. Biochemical systematics and Ecology 34: 97-113.

Moraes, SC & Vasconcelos, HL (2009) Long term persistence of a neotropical ant-plant population in the absence of obligate plant-ants. Ecology 90: 2375-2383.

Orivel, J & Leroy, C (2011) The diversity and ecology of ant gardens (Hymenoptera: Formicidae; Spermatophyta; Angiospermae). Myrmecological News 14: 73-85.

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ID number: XXXXX Authcate: xxxxx Assignment 1d: Scientific Literature Review Rosumek, FB, Silveirs, FAO, Neves, FS, Barbos, NPU, Diniz, L, Oki, Y, Pezzini, F, Femandes, GW & Comelissen, T (2009) Ants on plants: a meta-analysis of the role of ants as plant biotic defences. Oecologia 160: 537549.

Schatz, B, Djieto-Lordon, C, Dormont, L, Bessiere, JM, McKey, D & Blatrix, R (2009) A simple non-specific chemical signal mediates defence behaviour in a specialised anplant mutualism. Current Biology 19: 361-362.

Schmit-Neuerburg, V & Bluthgen, N (2007) Ant-garden epiphytes are protected against drought in a venezuelan lowland rain forest. Ecotropica 13: 93-100.

Servigne, P & Detrain C (2009) Seed desiccation limits removal by ants. Arthropod-plant Interactions 3: 225-232.

Vantaux, A, Dejean, A, Dor, A & Orivel, J (2007) Parasitism versus mutualism in the ant-garden parabiosis between Camponotus femoratus and Crematogaster levior. Insectes Sociaux 54: 95-99.

Vittecoq, M, Djieto-Lordon, C, Buatois, B, Dormont, L, McKey, D & Blatrix, R (2011) The evolution of communication in two ant-plant mutualisms. Evolutionary Biology 38: 360-369.

Weir, TL, Newbold, S, Vivanco, JM, Haren, MV, Fritchman, C, Dossey, AT, Bartram, S, Boland, W, Cosio, EG & Kofer, W (2012) Plant-inhabiting ant utilizes chemical cues for host discrimination. Bio Tropica 44: 246-253.

Youngsteadt, E, Nojima, S, Berlein CH, Schulz S & Schal, C (2008) Seed odor mediates an obligate antplant mutualism in Amazonian rainforests. Proceedings of The National Academy of Sciences 105: 4571-4575.

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