ISSN : 0917-415X DOI:10.3759/tropics.MS15-19

TROPICS Vol. 25 (3) 101-106 Issued December 1, 2016

ORIGINAL ARTICLE A bioassay for measuring the intensities of defenses on Macaranga myrmecophytes

Usun Shimizu-kaya1, 2*, Tadahiro Okubo2 and Takao Itioka2

1 Center for Ecological Research, Kyoto University, Hirano, Otsu, Shiga 520‒2113, Japan 2 Graduate School of Human and Environmental Studies, Kyoto University, Kyoto 606‒8501, Japan * Corresponding author: [email protected] Present address: Research Core for Interdisciplinary Sciences, Okayama University, Okayama 700‒8530, Japan

Received: January 10, 2016 Accepted: March 15, 2016 J-STAGE Advance published date: October 4, 2016

ABSTRACT In the Southeast Asian tropics, the tree Macaranga includes many myrmecophytic that associate with ‘plant-’ nesting in their domatia spaces. Plant-ants on Macaranga myrmecophytes protect their host-plants against herbivores. Because interspecific differences in ant defense intensities among Macaranga myrmecophytes affect the host-plant use by herbivorous , they need to be studied to better understand the ecology and evolution of herbivores on Macaranga myrmecophytes. In this study, to examine whether larvae of a lycaenid species, Arhopala major, which potentially feeds on some Macaranga myrmecophytes, can be used for a bioassay that assesses relative ant aggressiveness towards general herbivores on Macaranga plants, we experimentally introduced A. major larvae onto leaves of three Macaranga myrmecophytic species. We measured (1) the time required for the first touch by plant-ants on an introduced larva and (2) the number of plant-ant workers aggregating 3 min after the first touch. The order of three Macaranga species in ant defense intensity, as estimated by the two measurements, corresponded with the results of previous studies investigating the interspecific differences in ant defense intensities using ant-exclusion experiments. This suggests that the bioassay using A. major larvae is valid for the assessment of relative intensities of ant defenses on Macaranga species. As time and labor cost are low and standardization is easier, compared with the other methods, such as ant-exclusion experiments, the bioassay tested in this study is practical for assessing ant defense intensities on Macaranga plants.

Key words: ant-plant interactions, anti-herbivore defense, Arhopala major, Borneo, -plant interactions

INTRODUCTION with one, or a few, specific ant species (Fiala et al. 1999, Itino et al. 2001, Ueda et al. 2015). At least several Some plant species attract ants to use them as an anti- myrmecophytic species of Macaranga depend so strongly herbivore defense, termed ant defense (Beattie 1985, Heil on their plant-ants for their anti-herbivore defense that they and McKey 2003, Rico-Gray and Oliveira 2007, Ness et al. can hardly survive and grow without the symbioses with 2010). To date, approximately 4,000 plant species have their plant-ants (Fiala et al. 1994, Itioka et al. 2000). Itioka been reported to bear extra floral nectaries to attract ants et al. (2000) demonstrated that the intensities of ant (Weber and Keeler 2013) and more than 680 plant species defenses differ among a few of the myrmecophytic species are known as myrmecophytes or ‘ant-plants’, which have on Macaranga. Recently, a few herbivorous species were modified structures, so-called domatia, to host specific ant reported to specifically feed on a Macaranga species (plant-ants) (Chomicki and Renner 2015). To myrmecophytic species by evading the ant defense using evaluate the effectiveness of ant defense in a plant species, chemical and behavioral tactics (Okubo et al. 2009, it is essential to elucidate the associations between the plant Shimizu-kaya et al. 2013b, 2015, Inui et al. 2015, Shimizu- and the ant species (Bronstein 1998, Heil and McKey 2003, kaya and Itioka 2015). The narrow host-plant ranges of the Oliveira and Freitas 2004, Bronstein et al. 2006, Rico-Gray herbivores have been suggested to partly reflect the and Oliveira 2007, Del-Claro et al. 2013). interspecific differences in the ant defense intensities The tree genus Macaranga (Euphorbiaceae) contains (Shimizu-kaya et al. 2013a, Shimizu-kaya and Itioka 2015). approximately 25 myrmecophytic species that are To elucidate the ecological processes and the evolutionary distributed on the Malayan archipelago (Davies et al. 2001). adaptation of the herbivores that feed on Macaranga plants, Generally, a myrmecophytic Macaranga species associates including myrmecophytes, it is necessary to estimate the 102 TROPICS Vol. 25 (3) Usun Shimizu-kaya, Tadahiro Okubo et al. interspecific differences in the ant defense intensities Macaranga species that have been suggested to differ in ant properly. However, the differences remain unknown for defense intensity by Itioka et al. (2000) as determined by most Macaranga myrmecophytic species, except a few of other methods. Here, the practicality of the bioassay was the target species of Itioka et al. (2000). evaluated based on the measurement of the time required A major approach to measuring the effectiveness and/ for the plant-ants to detect the introduced larva and the or intensity of ant defense is the ant-exclusion experiment number of plant-ant workers aggregating around the larva 3 (these studies were listed in Chamberlain and Holland min after detection. We focused on whether the indices 2009, Rosumek et al. 2009). In this method, the correctly reflect the interspecific differences. effectiveness is estimated by measuring the increase in the abundance of, or damage by, herbivores when the plant-ants were experimentally excluded for various time periods, MATERIALS AND METHODS from a few weeks to several months (e.g. Fiala et al. 1994, Itioka et al. 2000, Heil et al. 2001, Dejean et al. 2006, Dutra Study site and materials et al. 2006, Queiroz et al. 2013). Although the ant-exclusion experiment is an appropriate way to clarify how ants benefit This study was conducted in a primary lowland mixed plants and influence other predators, it often takes time and dipterocarp forest in Lambir Hills National Park, Sarawak, causes serious damage to the tested plants. If it were Malaysia (4°13′N, 113°59′E, 50‒150 m asl). The site is possible to estimate ant defense intensities by directly located in the humid tropics with～2.6 m annual measuring ant aggressiveness when they are elicited by precipitation and a mean temperature of～27 ℃ (Kumagai herbivores, then the cost problems associated with et al. 2009). conducting ant-exclusion experiments could be minimized. At least 17 Macaranga species occur in the forest In fact, the intensities of ant defenses were estimated by (Davies 2001, Lee et al. 2002). We investigated the direct observation of ant behaviors in response to intensities of ant defenses in three myrmecophytic experimentally placed leaf discs, leaf extracts of the host Macaranga species, Macaranga winkleri Pax and K. Macaranga species, or to artificial leaf damage (Itioka et al. Hoffm., M. beccariana Merr. and M. trachyphylla Airy 2000, Bruna et al. 2004, Inui and Itioka 2007, Nomura et al. Shaw. The three species occur in open habitats such as 2011). However, it is difficult to standardize the size of riverbanks, forest edges, and gaps. Each of the three species discs and the use of leaf discs under different conditions, harbors one or a few specific ant species inside hollow such as microenvironments, age (size) of individual trees, stems: M. winkleri is associated with Crematogaster sp. 2, and the leaf positions within an individual plant, as well as M. beccariana is associated with C. decamera, and M. the manner of artificial leaf damage, for interspecific trachyphylla is associated with one or two species of the C. comparisons. borneensis group (Itino et al. 2001). In the three Macaranga In the present study, we tested the practicality of a type species, associations with plant-ants start as small seedlings of bioassay for measuring the intensities of ant defenses in with ant colonization (Fiala and Maschwitz 1992). At the myrmecophytic Macaranga species using the larvae of growth stage, plants start to produce food bodies on the Arhopala major Staudinger l889 (, ). abaxial surfaces of stipules or young leaves (Fiala and The larvae of A. major normally feed on non- Maschwitz 1992, Itioka et al. 2000) and extrafloral nectar myrmecophytic Macaranga species (Okubo et al. 2009), on the surfaces of young leaves (Fiala and Maschwitz but can grow to the adult stage by feeding on several 1991). Plant-ant workers collect food bodies and myrmecophytic Macaranga species under ant-excluded continuously patrol the plant surface. When plant-ant conditions (Shimizu-kaya et al. 2013a). Intensive ant workers find intruders such as herbivorous insects on the defenses on myrmecophytic Macaranga species might host plants, ants attack the intruders with their mandibles prevent the larvae of A. major from using host-plants in the and mass recruiting systems (Fiala et al. 1989). Then plant- field (Shimizu-kaya et al. 2013a). We hypothesized that the ants drop the intruders from the plants or force them to larvae of A. major may be potential herbivores of withdraw from the plants without feeding on them (Fiala et Macaranga myrmecophytes and thus, suitable to be used in al. 1989, Hashimoto et al. 1997). Although the plant-ants' the bioassay to measure ant aggressiveness toward defensive behaviors against the intruders are considerably herbivores. similar among the three plant-ant species, the intensities of In the bioassay tested here, A. major larvae were ant defenses are suggested to differ among the three species experimentally placed on to three myrmecophytic based on the results of three methods: ant-exclusion Bioassay for ant defense intensities on Macaranga 103 experiments, assessment of ant aggressiveness towards leaf the introduced larva to describe how long plant-ants take to tips, and measurement of the ratio of ant to tree biomass detect insect herbivores. At 3 min after the first touch, we (Itioka et al. 2000). It is suggested that the intensity of the counted the number of plant-ant workers aggregating ant defense is highest on M. winkleri and lowest on M. around the larva as an indicator of the intensity of defensive beccariana (Itioka et al. 2000). behavior by plant-ants towards insect herbivores. Each Larvae of A. major Staudinger, 1889 normally feed on tested tree and each larva were used for a single trial. young leaves of non-myrmecophytic M. gigantean (Rchb.f. and Zoll.) Müll. Arg. (Okubo et al. 2009), whereas they potentially feed on leaves of several myrmecophytic RESULTS Macaranga species, including M. winkleri, M. trachyphylla, and M. beccariana, under ant-excluded conditions The time until the first touch by plant-ants on an (Shimizu-kaya et al. 2013a). The larvae have three types of introduced larva (time for initial touch) was significantly myrmecophilous organs, the dorsal nectary, tentacle, and different among the three Macaranga species (Kruskal- pore cupola organs (Okubo et al. 2009). The leaves of host- Wallis test: P=0.00046; Fig. 1a). It was significantly shorter plants are attended by some ant species that are not specific on M. winkleri than on M. beccariana (Tukey post-hoc symbionts of any particular Macaranga myrmecophytes, HSD test: P=0.0087; Fig. 1a). The average time on M. while the frequency and intensity of ant attendance is much trachyphylla was approximately five times as long as that lower than in other Arhopala species found on on M. winkleri (Tukey post-hoc HSD test: P=0.17) and myrmecophytic Macaranga species (Okubo et al. 2009). approximately two thirds of that on M. beccariana (Tukey When a larva of A. major is experimentally placed on a leaf post-hoc HSD test: P=0.53). around the apical part of a myrmecophytic Macaranga On all of the tested trees, plant-ant workers aggregated species, plant-ants on the myrmecophyte bite off the larva and surrounded the introduced larva after the first touch. into pieces, or pull it towards the edge of the leaf, to drop it The aggregating ant workers bit the introduced larva from the tree (Shimizu-kaya, unpublished data). Based on persistently. The number of ant workers aggregating at 3 our observation of the plant-ants’ attacking behaviors, the min after the first touch was significantly different among plant-ants are inferred not to feed on A. major larvae, or the the three Macaranga species (Kruskal-Wallis test: amount of materials and nutrients that they might intake as P<0.0001; Fig. 1b). It was significantly higher on M. food from the larvae bodies would be very limited even if winkleri than on M. trachyphylla and on M. beccariana they feed on the larvae. It remains to be determined whether (Tukey post-hoc HSD test: P=0.0039 and P<0.0001, plant-ants feed on any larvae during the process. respectively; Fig. 1b). The average number of ants on M. trachyphylla was more than twice that on M. beccariana (Tukey post-hoc HSD test: P=0.089). Larval introduction experiments

These experiments were conducted from February to DISCUSSION March 2007, from July to August 2011, and in July 2015. We collected larvae of A. major from M. gigantean in the In the bioassay, the time until the first touch by plant- field and kept them for a few days in the laboratory on M. ants on an experimentally introduced larva and the number gigantean leaves in plastic boxes (20×15×10 cm) until of aggregating ant workers after the first touch are thought they reached middle or semi-final instar stages. The M. to reflect the plant-ants’ sensitivity and aggressiveness to gigantean leaves used for larval feed were collected the intrusion of insect herbivores. These properties of plant- haphazardly from individual trees in the field. We selected ants towards insect herbivores are considered to strongly 11 trees of M. winkleri, 12 trees of M. trachyphylla, and 10 correlate with the total intensity of anti-herbivore defense trees of M. beccariana with heights ranging from 1.2 to on the plants via the plant-ants. Our results indicate that 2.0 m. We confirmed that all of the trees harbored plant-ants insect herbivores would be detected more quickly and and did not bear any obvious herbivores, or noticeable attacked more intensely by plant-ants on M. winkleri than damage. On the main vein of the abaxial side of the second on both M. trachyphylla and M. beccariana, and on M. apical leaf of each selected tree, we centered a single larva trachyphylla than on M. beccariana. The rank order of the using a wet paintbrush quickly without stimulating plant- intensities of ant defenses on the three Macaranga species ants. We recorded the time when a plant-ant first touched shown by the results of the bioassay is consistent with the 104 TROPICS Vol. 25 (3) Usun Shimizu-kaya, Tadahiro Okubo et al.

intensities of ant defenses on Macaranga myrmecophytes for the following reasons: First, the plant-ants’ aggressive behaviors towards A. major larvae on a Macaranga myrmecophyte is considered to represent the plant-ants' response to various types of herbivores that can feed on the myrmecophyte in the field because A. major is also one of the various herbivores that can feed on a wide range of Macaranga myrmecophytes (Shimizu-kaya et al. 2013a). Second, A. major larvae are relatively abundant in the area where Macaranga myrmecophytes are distributed. The host-plant species M. gigantean occurs in broad range of habitats in the area (Davies 2001). In addition, they are usually motionless on the plants. This is useful for the manipulation and standardization of the bioassay. In addition to the interspecific differences in intensities of ant defenses on Macaranga myrmecophytes, it was expected that intraspecific differences could occur owing to differences in associated plant-ant species (Maschwitz et al. 1996, Feldhaar et al. 2003, Murase et al. 2003), environmental conditions in the habitats, and influences of disturbances (Shimizu-kaya et al. 2015). Moreover, ant defense intensities may vary between young and mature parts (Heil et al. 2004, Queiroz et al. 2013), or the developing and mature stages (Itino and Itioka 2001, Handa et al. 2013) within a plant individual. These variations should affect host-plant use and feeding habits of herbivorous insects. The bioassay methods will be applied Fig. 1. Two indicators of ant defense intensities on three in future studies investigating the ant-plant-herbivore Macaranga myrmecophytes, M. winkleri, M. trachyphylla, and M. beccariana. (a) Time until the first interactions on Macaranga myrmecophytes. touch by plant-ants on an experimentally introduced Arhopala major larva and (b) number of plant-ants aggregating around the larva 3 min after the first touch. ACKNOWLEDGEMENTS The fieldwork in Lambir Boxplots show the ranges, quartiles, and averages. Hills National Park was conducted in accordance with the Different letters indicate significant differences among the three Macaranga species (Kruskal-Wallis with post Memorandum of Understanding signed by the Sarawak hoc tests, p<0.01). Forestry Corporation and the Japan Research Consortium for Tropical Forests in Sarawak (JRCTS) in November 2005, and the Memorandum of Understanding signed by rank order estimated from the results of previous studies by the Sarawak Forest Department and JRCTS in November Itioka et al. (2000) and Nomura et al. (2011). Thus, the 2012. We thank M. S. Sabki, M. Kohdi, P. Meleng and F. bioassay using A. major larvae appears to be practical for Mohammad of the Sarawak Forest Department for the help estimating the intensities of ant defenses on Macaranga in obtaining permission to conduct this study. We also thank myrmecophytes. The differences in the two traits between T. Nakashizuka (Tohoku University, Japan), N. Yamamura M. trachyphylla and M. beccariana seem not to be (Doshisha University, Japan), and S. Sakai (Kyoto significant. However, the more intensive ant defense on M. University) for supporting our research. This study was trachyphylla than on M. beccariana is consistent, as funded by Grants-in-Aid to T.I. from the Japan Society for determined by all of the types of comparisons used to assess the Promotion of Science (No. 21255004) and by the the two traits, although we were not able to procure Research Institute for Humanity and Nature, Japan (No. sufficient plant samples for robust statistical analyses. D-04). Compared with the other insect herbivores, A. major larvae are considered to be more suitable for measuring the Bioassay for ant defense intensities on Macaranga 105

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