Host-choice

5.1. INTRODUCTION

While most Cladomyrma species appear to have a restricted host range, that of the genus as a whole is broad. Host affiliation shows considerable variation both at plant species and genus level (Tab. 11-1 Fig. 11-1 e.g., ant species/plant genus pairings range from 1:1 for Cladomyrma maschwitzi (Crypteronia) to 1:6 for C. petalae (Saraca, Spatholobus, Drypetes, Ryparosa, Luvunga, and Strychnos). The latter species has a very broad host plant spectrum, comprising unrelated host species from five different families. At Fraser’s Hill, Malay Peninsula, five host plant species occur sympatrically in a narrow elevational band at 800 m a.s.l. (Drypetes, Luvunga, Ryparosa, Saraca, Spatholobus). Along one transect of only 50 m these hosts were all found to be inhabited by C. petalae.

This surprisingly broad host spectrum poses a distinct adaptive problem. What are the signals involved in host-finding and host-choice by founding ant-queens? It appears highly unlikely that these unrelated hosts possess similar scent profiles differentiating them against a diverse background of plants. It could be argued that Cladomyrma petalae is a ‘jack-of-all-trades’, colonising any suitable plant in the vicinity of its natal host. Localisation of hosts would then be independent of taxon, instead the detection of host could be determined by general traits such as age of plant tissue and internode size only. Unspecific volatile organic compounds emitted by young plant tissue may act as a general signal to foundress queens of Cladomyrma petalae (DICKE 2000, MÜLLER & HILKER 2000, PICHERSKY & GERSHENZON 2002).

This scenario, however, is not the case. Although I cannot rule out that host range might be still more diverse than is currently known, both observations in the field and the examination of nearly 5,000 herbarium specimens of congeneric host taxa indicate that host plants are not unspecifically selected by the ant queens.

Under these circumstances, which mechanisms are involved in the restriction of Cladomyrma petalae to only a few, unrelated hosts? Host-choice may be based on a learned odor profile. Foundress ant queens search for individuals of the plant species in which they were raised. In the obligate ant-plant system Barteria-Tetraponera it has been demonstrated that experience gained during the larval and nymphal stages as well as during the first part of adult life had a (weak) effect on nest plant selection by ant workers (DJIETO-LORDON & DEJEAN 1999). However, innate attraction of Tetraponera aethiops workers to the host Barteria fistulosa remained strong, even

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when workers were raised in the presence of non-Barteria test plants. The authors conclude that under natural conditions the influence of the environment reinforces innate attraction.

Methodological problems make bioassays for testing host-choice of plant-ant queens notoriously difficult. First, it is impossible to use naïve queens for studies, because they cannot be raised apart from their hosts. Second, it is almost impossible to catch alate queens directly after mating but before colony-founding. Even when successful the natal host would then be unknown. Third, the use of virgin alate queens collected from the natal host prior to the nuptial flight may result in non-natural responses due to lack of insemination or other possible cues (e.g., flight distance before colony-founding). Fourth, queens may be collected during the process of colony-founding but these queens have already shed their wings. When used in an experiment they have a reduced mobility and thus are probably under pressure to make a choice even if the host plant candidate appears less suitable.

Furthermore, collecting colony-founding queens will only produce a low sample size due to the high search effort. Lastly, nulliparous queens (that have not yet laid eggs) can be collected from internodes of freshly colonised plants (e.g., INUI et al. 2001) but the previous and presumably exhaustive task of colony-founding may influence the subsequent behaviour of the queens in host-choice experiments in unknown directions.

Considering the methodological problems involved in bioassays on host-choice I decided to test whether foundress queens of Cladomyrma petalae accept other sympatric hosts or non-host plants. Are the queens capable of accepting sympatric hosts despite their phylogenetic distance to the source plant? Are they forced to enter non-host species when having no choice? In a wider context: What is the level of plasticity in the choice of the supporting plant? Is there a potential for shifting to new hosts on an individual level?

In the following, I will present some preliminary results of no-choice tests on host acceptance by Cladomyrma petalae queens.

5.2. MATERIAL AND METHODS

The test were conducted using branches (n=21) with young internodes and suitable stem diameter of six different plant species known to be hosts of Cladomyrma petalae.

Branches with fresh domatia of an allopatric ant-plant from Sumatra, Neonauclea cyrtopoda, served as control (n=10). A bushy tree was kept in a greenhouse at the study site in Ulu Gombak, West-Malaysia. N. cyrtopoda is regularly inhabited by an obligate Crematogaster species and thus generally suitable for ant-inhabitation. I do

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not expect it to possess hidden traits deterring ants. The latter holds true for the sympatric, non-specific ant-tree Pometia pinnata forma glabra (MOOG et al. 2008).

Young branches were also used in the bioassay as controls (n=4).

Foundress queens of C. petalae in the process of colony-founding (gnawing a hole into the stem) were searched and collected from plants of Saraca thaipingensis, Spatholobus bracteolatus and Strychnos vanprukii (source plants). All these queens had already shed their wings.

The test branch was placed upright in the middle of a plastic box surrounded by water, preventing the queens to leave the arena. Then, a foundress queen was introduced at the apex of the branch. The behaviour of each queen was monitored for one hour. A plant was considered as ‘accepted’ when the queen had clearly left a deep mark on the plant surface by chewing.

Gnawing a hole into the stem is the last step in the process of host-selection behaviour (Fig. 5-1). Foundresses that did not show any sign of the typical stereotyped founding behaviour during the observation period were placed back onto the source plant. If, then, the queen accepted the original source plant I considered the previous rejection as valid. However, if a queen did not return to normal foundation behaviour on the source plant the rejection of the former plant was considered invalid and the data were omitted from the analysis. This design reduces effects on queen behaviour by handling or other possible disturbances. Each queen was tested only once.

Figure 5-1: Colony-founding by Cladomyrma petalae. A dealate queen chews a hole into a young internode of Saraca thaipingensis.

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5.3. RESULTS

The results are summarised in table 5-1. Foundress queens of Cladomyrma petalae accepted one third (7 of 21 trials) of the offered sympatric host-plants, whereas control plants were always refused (0/14). The difference between sympatric hosts and controls is significant (Fisher’s exact test, P = 0.0272, df = 1, chi square 5.833).

A more detailed analysis of the data would reveal that several typical steps in the host-selection behaviour ¹ of Cladomyrma were observed in the test plants of sympatric hosts – even if they were not accepted within the observation period of one hour.

These behavioural responses (see footnote step ii or iii to v) of the queens were usually not observed in the controls. Moreover, it should be noted that a certain percentage of ‘correct’ host plant species is rejected even under natural conditions. In the field I noticed three winged queens which, after a thorough inspection of young Saraca stems (taking 2–14 min), flew off and continued their search.

Table 5-1: Host-acceptance experiment with queens of Cladomyrma petalae (no-choice test).

Foundress queens collected during the founding process from source plants were introduced to young branches of sympatric hosts and controls. Given are numbers of accepted plants; total number of tested plants in parenthesis (). For details see text.

experimental plant\source plant: Saraca

1These steps include: (i) running in a more or less straight line up and down the internode; (ii) changing to a sinusoidal running curve, thereby encircling the internode from time to time, sometimes entering the adjacent leaf but returning to the stem; (iii) stopping now and then for several seconds to inspect a spot on the internode; (iv) slowing down the movements and extending the inspection time of stem spots, typically accompanied by antennating, palpating and test biting; (v) gnawing a hole into the stem (see also MASCHWITZ et al. 1991).

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5.3.1. Summary of the results: host-choice

• queens of Cladomyrma petalae, a species with a taxonomically diverse host range, can accept sympatric hosts when having no choice

• plants not belonging to the ‘usual’ host spectrum are not accepted

5.4. DISCUSSION

This study suggests that foundress queens of Cladomyrma petalae possess some plasticity in host-choice. Under experimental conditions queens can be ‘forced’ to shift to other plants of their ‘usual’ host spectrum despite their phylogenetic distance. Non-host plants appear to be consistently refused but sample size is low and a more detailed study is required. Rejection of a plant is fatal for dealate queens since they can hardly find a new suitable host within ‘walking distance’.

Does a foundress queen of Cladomyrma petalae select sympatric plants of the host spectrum other than their natal host under natural conditions? Indirect and circumstantial evidence comes from observations of climbing hosts in the field. Two liana species, Strychnos vanprukii and Spatholobus bracteolatus were found to climb sympatric host trees, Saraca thaipingensis and Drypetes longifolia, respectively. In each case (n=4), both the climber and the supporting tree harboured a single ant colony of Cladomyrma petalae, with the queen located either in the stem of the tree (n=3) or inside the climber (n=1). The workers of each colony had gnawed fresh nest chambers into young internodes of both partner species. This colonisation pattern suggests that the workers are capable of recognising host plants of the ‘usual’ host spectrum. [Note that non-host lianas climbing Cladomyrma host trees are, of course, never inhabited.]

Queens alighting on a host and checking internodes for their suitability as nest site presumably use several cues for host recognition (vision, olfaction and contact chemoreception). It is highly unlikely that the unrelated hosts show similarities in general stimuli such as whole scent profiles. In an ant/plant system involving species of a single genus only (Macaranga), odor profiles showed distinct interspecific variation (JÜRGENS et al. 2006). Even Macaranga species sharing the same obligate ant morphospecies (Crematogaster msp. 1, 2, 4) were found to show no obvious similarities in their scent profiles. The authors assume that ant queens may rely only on ‘key stimuli’, a few or a combination of several leaf compounds, for the choice of their specific host species.

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In the case of the African tree Leonardoxa africana, ants of Petalomyrmex phylax workers are attracted by ‘green-leaf volatiles’ and methyl salicylate, which is emitted in high levels only by the young leaves (BROUAT et al. 2000). These compounds are reported as common components of numerous plant species belonging to a variety of plant families, and they are usually released from plants after herbivore attack (HEIL

2008). BENSON (1985) suggested that ant-plant symbioses originated from ants tending coccoids on plants. The ants might originally have used wound induced plant volatiles to find hosts infested with coccoids, as do parasitoid wasps and predaceous insects (CORTESERO, STAPEL & LEWIS 2000). Over evolutionary time, as a facultative plant mutualism evolved into an obligate one, it has been hypothesised that ant-plants evolved to release ant-attracting volatiles even in the absence of herbivory, thus insuring worker patrolling of vulnerable plant parts and the encounter of host plants and foundress queens (BROUAT et al. 2000, EDWARDS et al. 2006).

While the precise nature of plant-derived signals are not yet clear, exaptations –pre-existing traits that acquired new functions– could also explain host plant recognition in the Cladomyrma system. Host plants of diverse phylogenetic affiliations may possess common substances related to herbivory (e.g., green-leaf volatiles and methyl salicylate). Driven by evolutionary interaction with their mutualistic ants, Cladomyrma host plants might have specialised in producing high concentrations of such compounds from intact young plant tissue, thus mimicing signals emitted by wounded leaves. Attraction of ant workers to damaged leaves has been demonstrated in a variety of ant/plant systems (e.g., FIALA & MASCHWITZ 1990, AGRAWAL & DUBIN-THALER

1999, LAPOLA, BRUNA & VASCONCELOS 2003, ROMERO & IZZO 2004, INUI & ITIOKA

2007).

In some cases, the compounds emanating from ant-plants have been found to be similar or identical to semiochemicals produced by the ants themselves. Some green-leaf volatiles, namely hexanal and 1-hexanol, of two myrmecophytic Piper species (MAYER, SCHABER & HADACEK 2008) are major components of the mandibular glands of major workers of Oecophylla longinoda. These chemicals cause an alarm and attraction response when presented to foraging workers (BRADSHAW, BAKER & HOWSE

1975). Another volatile organic compound of the two Piper ant-plants, the sesquiterpene "-caryophyllene, has been shown to evoke atagonistic behaviour in a leaf-cutting ant (NORTH, HOWSE & JACKSON 2000). Furthermore, methyl salicylate is similar to a compound produced by some ant species, methyl-6-methyl salicylate, which appears to be an alarm pheromone in some Ponerinae and a sex pheromone in Camponotus (DUFFIELD & BLUM 1975, HÖLLDOBLER & WILSON 1990).

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The most accurate and reliable information, however, about host identity and suitability is usually gained via contact chemoreception (HEISSWOLF et al. 2007). On the plant surface, plant cuticular waxes can already give important information for host plant acceptance (MÜLLER & RIEDERER 2005). Typical behaviours before acceptance of a host are antennating, palpating, and test biting; behavioural patterns that are also observed in Cladomyrma petalae foundress queens during host assessment. To sum up, host recognition and acceptance by Cladomyrma might be achieved through qualitative or quantitative blends of host volatiles acting in concert with contact stimuli of the plant surface. This example shows the potential of the Cladomyrma/plant system as model for exploring the flow of information between mutualistic partners and for investigating the mechanisms involved in the restriction of many plant-ants to one or only a few host(s).

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