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(American Journal of Botany. 2004;91:1319-1325.)
© 2004 Botanical Society of America, Inc.

Reproductive Biology

Obligate pollination mutualism in Breynia (Phyllanthaceae): further documentation of pollination mutualism involving Epicephala moths (Gracillariidae)1

Atsushi Kawakita2 and Makoto Kato

Graduate School of Human and Environmental Studies, Kyoto University, Yoshida-Nihonmatsu-cho, Sakyo, Kyoto 606-8501, Japan

Received for publication December 2, 2003. Accepted for publication May 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
This paper reports obligate seed-parasitic pollination mutualisms in Breynia vitis-idea and B. fruticosa (Phyllanthaceae). The genus Breynia is closely related to Glochidion and Gomphidium (a subgenus of Phyllanthus), in which pollination by species-specific, seed-parasitic Epicephala moths (Gracillariidae) have been previously reported. At night, female Epicephala moths carrying numerous pollen grains on their proboscises visited female flowers of B. vitis-idea, actively pollinated flowers, and each subsequently laid an egg. Examination of field-collected flowers indicated that pollinated flowers of B. vitis-idea and B. fruticosa almost invariably had Epicephala eggs, suggesting that these moths are the primary pollinators of the two species. Single Epicephala larvae consumed a fraction of seeds within developing fruit in B. vitis-idea and all seeds in B. fruticosa. However, some of the fruits were left untouched, and many of these had indication of moth oviposition, suggesting that egg/larval mortality of Epicephala moths is an important factor assuring seed set in these plants. The overall similarity of the specialized floral structure among Breynia species may indicate that this pollination system is fairly widespread within the genus.

Key Words: Breynia • Epicephala • Glochidion • Gracillariidae • obligate pollination mutualism • Phyllanthaceae • Phyllanthus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The classically known obligate pollination mutualisms between figs–fig wasps and yuccas–yucca moths are among the most fascinating examples of pollination mutualisms known (Janzen, 1979 ; Weiblen, 2002 ; Pellmyr, 2003 ). In these systems, figs and yuccas depend exclusively on adult female wasps and moths for pollination, respectively, while the adult females depend on the developing seeds for nourishment of their offspring. These mutualisms are unusual both in the diversity of species involved and host specificity of the pollinators (Pellmyr, 1999 ; Weiblen, 2002 ; Molbo et al., 2003 ). In addition, these interactions involve highly coevolved traits, such as active pollination behavior and specialized floral structures, topics of general biological interest (Kjellberg et al., 2001 ; Pellmyr and Krenn, 2002 ; Jousselin et al., 2003 ). Furthermore, costs and benefits of the interaction for the plant (seed production and seed consumption) are relatively easy to measure, thus facilitating ecological analysis of the outcome of mutualism in these plants (Addicott, 1986 ; Pellmyr and Huth, 1994 ; Herre and West, 1997 ; Addicott and Bao, 1999 ; Patel and Hossaert-McKey, 2000 ). Together, these attributes of the fig–fig wasp and yucca–yucca moth interactions provide model systems for various analyses of coevolutionary processes and ecological dynamics of mutualism.

The recently discovered obligate pollination mutualism between Epicephala moths and trees of the family Phyllanthaceae (formerly Euphorbiaceae; see APG, 2003 ) possesses striking similarities with the fig–fig wasp and yucca–yucca moth systems and potentially provides a model system for studies of coevolution and mutualism (Kato et al., 2003 ; Kawakita and Kato, 2004 ). In these associations, trees of the genera Glochidion and Phyllanthus (subgenus Gomphidium) are pollinated exclusively by the females of species-specific Epicephala moths that actively collect and transport pollen with their proboscises. The moths lay eggs in female flowers, and their offspring consume 28–74% of the developing seeds while leaving the rest intact (Kato et al., 2003 ; Kawakita and Kato, 2004 ). These associations are extremely diverse with Glochidion and Gomphidium together comprising more than 450 species (Govaerts et al., 2000 ), while high host specificity of the pollinator moths indicate that a comparable number of Epicephala moths also exist (Kato et al., 2003 ; Kawakita and Kato, 2004 ). In addition, there are multiple sources of variation in modes of the plant–moth interaction (e.g., difference in the number of ovules per fruit; partial or total destruction of fruit by a single moth larva; Kato et al., 2003 ; Kawakita and Kato, 2004 ), allowing comparative approaches for the studies of evolutionary and ecological dynamics of the mutualism.

While previously reported mutualisms between Glochidion/ Gomphidium and Epicephala provide novel opportunity for studies of pollination mutualisms and coevolutionary processes, the pollination systems in closely related plant genera have yet to be investigated. Here, we provide evidence for obligate pollination mutualism in two species of Breynia (B. vitis-idea and B. fruticosa), which are closely related to Glochidon and Gomphidium (Webster, 1994 ; Govaerts et al., 2000 ). The genus Breynia comprises 35 species of monoecious shrubs, distributed in tropical and subtropical regions of Asia, Australia, and the Pacific Islands (Webster, 1994 ; Govaerts et al., 2000 ). In this paper, we determine whether Epicephala moths associated with Breynia plants constitute the primary pollinators in the two species. We also investigate if there is variation in the cost of mutualism (i.e., seeds consumed by pollinator larvae) between the two species, which potentially affects the outcome of the interaction. Finally, we compare our results with those of the Glochidion and Gomphidium systems and discuss major variants in the mode of interaction within the Phyllanthaceae– Epicephala mutualism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Breynia vitis-idea
Breynia vitis-idea is a monoecious shrub that occurs in forest margins of tropical and subtropical forests in Asia (Fig. 1). The species is distributed from Pakistan to the southern part of Japan, including most parts of tropical Southeast Asia (Govaerts et al., 2000 ). The flowers lack petals and are dimorphic, with male flowers arranged toward the base and female flowers at the apex of each branch (Fig. 2). Typically, only one or two flowers are born on axils. Male flowers have fused calyx lobes with inflexed apical ends that make the stamens unlikely to be accessible to opportunistic flower visitors (Fig. 3). Female flowers are campanulate with three short styles fused at the center of the upper surface of the ovary (Fig. 4). Female flowers have three locules, each containing two ovules. Fruits are produced shortly after pollination within 3–4 wk. In the course of fruit development, pedicels become erect, and the fruit coat eventually turns red to dark purple (Fig. 5). Flowering and fruiting occur throughout the year but typically peak in spring (March to May) and early fall (August to October) at our study sites.



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  		Figs. 1–14. Flowers, fruits, and associated insects of Breynia vitis-idea and B. fruticosa. 1. B. vitis-idea plant. 2. Branch of B. vitis-idea with male flowers toward base (to left) and female flowers toward apex (right) 3.–5. Breynia vitis-idea. 3. Male flowers. 4. Female flowers. 5. Fruits. One of the fruits has an exit hole of Epicephala moth larva (arrowhead). 6.–8. Breynia fruticosa. 6. Male flowers. 7. Female flowers. 8. Fruits. 9. Female Epicephala moth actively pollinating a B. vitis-idea female flower with its proboscis. 10. Female Epicephala moth ovipositing in a female B. vitis-idea flower. 11. Female Epicephala moth collected from a B. vitis-idea flower. Proboscis of moth is covered with pollen grains (arrowhead). Bar = 1 mm. 12. Cross section of B. vitis-idea fruit. The fruit has six seeds, three of which were destroyed by an Epicephala larva. The larva had emerged through the exit hole (arrowhead). Bar = 1 mm. 13. Female braconid wasp probing a B. vitis-idea fruit with its ovipositor. 14. Female Epicephala moth collected from a B. fruticosa plant having a pollen-coated proboscis (arrowhead). Bar = 1 mm

 
We studied insect flower visitors of B. vitis-idea during 27 September–1 October 2002 and 9–13 May 2003 at Kasari, Amami Island (28°28' N, 129°41' E), 29 September–2 October 2003 at Banna, Ishigaki Island (24°22' N, 124°10' E), and 3–5 October 2003 at Funaura, Iriomote Island (24°24' N, 123°48' E), Japan. Preliminary observations on flowering and fruiting phenologies were made at various localities in southern Japan in 2001–2003. We made diurnal and nocturnal observations of flower visitors for a total of more than 60 h during the study periods. Particular effort was made for nocturnal observations to study flower visitation by Epicephala moths. Epicephala moths that visited flowers were collected after they became inactive on branches or leaves. We also collected other insect visitors after they left the flowers. The collected insects were identified and examined for pollen attachment with a light microscope.

Because flowers of B. vitis-idea produced a small amount of nectar, we determined whether nectar production occurred during the day or at night using 10 marked female flowers on each of four individual plants. We covered the marked flowers with fine netting (0.25-mm mesh; Wataya, Kyoto, Japan) to exclude nectar foragers and sampled nectar at 0600 hours and 1800 hours during 11–13 May 2003 using microcapillaries (Drummond, Broomall, Pennsylvania, USA). We also measured sugar concentration of sampled nectar using a pocket refractometer (Bellingham & Stanley, Kent, UK) to monitor temporal changes in sugar concentration of nectar. We did not use male flowers for nectar measurements because of the difficulty of sampling nectar from enclosed male flowers without severe destruction to the flowers.

After field studies, we collected female flowers of B. vitis-idea, counted the number of pollen grains on stigmas, and examined the presence or absence of Epicephala moth eggs in flowers under a light microscope. Totals of 179 and 274 female flowers were sampled from three and four individuals at Kasari on 15 May 2003 and at Banna on 8 October 2003, respectively. To determine the extent of seed infestation by seed-parasitic moths, we also sampled 365 and 39 mature fruits from seven and two individuals at Kasari on 15 May and Funaura on 5 October 2003, respectively. For each fruit, we counted the number of destroyed seeds, intact seeds, and unfertilized ovules. In addition, whenever uninfested fruits were encountered, we looked for remains of Epicephala moth eggs to infer whether the moths had oviposited on the fruits. For the fruit samples collected at Kasari, we also assessed the cause of seed destruction for each fruit. Seeds were primarily destroyed by Epicephala larvae, but immature larvae were occasionally parasitized by a braconid wasp, and a nonpollinating carposinid moth, Paramorpha sp., also infested the seeds. We therefore determined the causes of seed destruction based on differences in the structure of feces and exit holes left by the insects. Also, the number of moths and braconid wasps that occupied each fruit was determined based on the number of exit holes on the surface of each fruit. Preliminary rearing of moths and wasps has indicated that these insects always bore an exit hole upon leaving the fruit; thus, the number of exit holes can be reliably used to estimate the number of insects emerged.

Breynia fruticosa
Breynia fruticosa is a monoecious shrub that is typical in forest margins of Indochina and southern China (Govearts et al., 2000 ; van Welzen et al., 2000 ). Typically, 2–4 flowers are born on each axil with male flowers arranged towards the base of each branch. Male flowers have fused calyx lobes with inflexed apical ends as those of B. vitis-idea (Fig. 6). Female flowers are not as specialized as in B. vitis-idea and have free calyx lobes and free styles (Fig. 7). The styles are split in the upper half and are likely accessible to opportunistic flower visitors (Fig. 7). Female flowers have three locules, each containing two ovules. Once pollinated, female flowers become erect, and fruits are produced within 3–4 wk after pollination (Y. Kosaka, Kyoto Univeristy, Japan, personal communication) (Fig. 8). Flowering and fruiting occurs year-round with several peaks per year (Y. Kosaka, personal communication).

We studied insect flower visitors of B. fruticosa during 9–11 March and 14–16 September 2003 at Dongmakhai (17°58' N, 102°36' E) and 17–18 September 2003 at Thakhaek (17°24' N, 104°48' E), Laos. We studied diurnal and nocturnal flower visitors for a total of 26 h during the study period with emphasis on nocturnal observations of Epicephala moths. All insect visitors were collected and examined for pollen attachment as described. In addition, we collected Epicephala moths that rested on leaves to check for pollen grains on their proboscises. After field observations, we collected totals of 210 flowers and 43 fruits from six B. fruticosa individuals to study pollen load on stigmas, presence or absence of Epicephala moth oviposition, and extent of seed destruction by moth larvae as described for B. vitis-idea. For fruits that were not infested by Epicephala moths, we looked for indications of moth oviposition (i.e., oviposition scars) to infer whether the Epicephala moths had oviposited on fruit. Flowers and fruits were collected at Dongmakhai on 20 September 2003.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Breynia vitis-idea
Nectar was produced at night on female flowers of B. vitis-idea (Table 1). The marked differences in the amount of nectar produced during the two nights was likely from the difference in air humidity during the two days (humid and dry on 12 and 13 May, respectively), which was reflected in sugar concentration of nectar (Table 1).


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  		Table 1. The amount and sugar concentration of nectar produced by female Breynia vitis-idea flowers during 11–13 May 2003. Values are means ± SE across four plant individuals (average value of 10 flowers for each plant)

 
Observed visitors to flowers of B. vitis-idea were the ant Anoplolepis longipes and an undescribed species of Epicephala moth. Workers of A. longipes visited female flowers of B. vitis-idea during the day but mainly at night to forage nectar. However, these ants were not observed on male flowers, and none carried pollen grains (N = 19). At night, we observed female Epicephala moths visiting female B. vitis-idea flowers, depositing pollen grains with their proboscises (Fig. 9) and subsequently laying an egg within the interspace between clayx lobes and ovary (Fig. 10). We observed four female Epicephala moths visiting female flowers, which all had the same stereotypic behavior on flowers. In addition, one of these moths repeated the pollination–oviposition behavior twice on the same flower. Although we did not observe Epicephala moths collecting pollen on male flowers, all the moths that visited female flowers carried numerous pollen grains on their proboscises (Fig. 11). The low frequency of moth visits reflects the rare occurrence of moth visitation and the short time Epicephala moths spent on flowers, which was also the case in Glochidion- and Gomphidium-pollinating Epicephala moths (Kato et al., 2003 ; Kawakita and Kato, 2004 ; A. Kawakita and M. Kato, personal observations).

Examination of pollen load and Epicephala moth eggs in female flowers revealed that nearly all pollinated flowers had moth eggs, whereas unpollinated flowers only rarely had eggs (Fig. 15). The mean number of pollen grains on female flowers with moth eggs was 12.0 ± 0.6 grains (mean ± SE; N = 116) and 9.8 ± 0.9 grains (N = 38) at Banna and Kasari, respectively, which was significantly greater than that on female flowers without moth eggs (0.4 ± 0.2 grains, N = 158 and 0.1 ± 0.1 grains, N = 141, Mann-Whitney U test; U = 409 and 23, P < 0.0001). These data indicate that Epicephala moths are likely exclusive pollinators of B. vitis-idea. Pollen grains were aggregated at the stigmatic part of female flowers as in Glochidion and Gomphidium (Kato et al., 2003 ; Kawakita and Kato, 2004 ), which is unlikely to occur through passive pollination. Eggs were laid between the ovary and calyx lobes, and on average, egg-loaded flowers had 1.58 and 1.53 eggs per flower at Banna and Kasari, respectively (N = 116 and 38, range: 1–4).



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  		Fig. 15. Frequency distributions of the number of pollen grains attached to stigmas of female Breynia vitis-idea flowers with and without Epicephala moth eggs at Banna and Kasari, Japan. The mean number of pollen grains attached to flowers with and without moth eggs was 12.01 ± 0.56 grains (mean ± SE; N = 116) and 0.37 ± 0.16 grains (N = 158) at Banna and 9.76 ± 0.93 grains (N = 38) and 0.11 ± 0.07 grains (N = 141) at Kasari, respectively

 
The mean number of intact seeds per fruit was 2.4 ± 0.1 seeds (N = 365) and 3.7 ± 0.4 seeds (N = 39) at Kasari and Funaura, respectively, and seed-parasitic moths destroyed 3.0 ± 0.1 and 2.3 ± 0.4 seeds (Table 2). However, 30.1% of the fruits sampled at Kasari and 43.6% at Funaura were not infested by the moths (Table 2). Of these uninfested fruits, 43.6% and 64.7% had remains of Epicephala moth eggs (Table 2), indicating egg/larval death of Epicephala moths in these fruits. Seed destruction was mainly caused by Epicephala larvae (Fig. 16). Normally, a single moth larva did not consume all seeds within a fruit (Figs. 12, 16), but two moth larvae were enough to destroy all seeds of a fruit (Fig. 16). Braconid wasps parasitized early instar Epicephala larvae by probing the fruit (Fig. 13); this parasitism had a significant positive effect on seed set by preventing further seed consumption by Epicephala larvae (Mann-Whitney U test; U = 135, P < 0.0001; Fig. 16).


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  		Table 2. Patterns of seed infestation by seed-parasitic moths in Breynia vitis-idea at two locations in Japan

 


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  		Fig. 16. Frequency distributions of the number of destroyed seeds per fruit in Breynia vitis-idea at Kasari. Fruits of B. vitis-idea invariably have six ovules. Immature larvae of Epicephala moths were occasionally parasitized by braconid wasps. The mean number of seeds destroyed was (A) 3.30 ± 0.11 seeds (mean ± SE; N = 83), (B) 5.66 ± 0.06 seeds (N = 119), (C) 1.48 ± 0.09 seeds (N = 31), and (D) 3.02 ± 0.13 seeds (N = 365)

 
Breynia fruticosa
Although we did not observe Epicephala moths visiting flowers in B. fruticosa, Epicephala females collected on leaves of B. fruticosa carried numerous pollen grains on proboscises as those pollinating B. vitis-idea (Fig. 14), which indicates that these moths are active pollinators. We also observed a gall midge, Clinodiplosis sp., resting at the entrance of male flowers or on the styles of female flowers. Of the 15 midges that we collected, two had a few pollen grains on legs and heads, suggesting that these gall midges may also contribute to pollination.

The pattern of relationship between pollination and Epicephala oviposition was similar to that observed in B. vitis-idea (Fig. 17), indicating that Epicephala moths are the primary pollinators of B. fruticosa. The mean number of pollen grains on female flowers with moth eggs was 14.6 ± 1.0 grains (N = 141), significantly greater than that on flowers without moth eggs (1.1 ± 0.4 grains, N = 69, Mann-Whitney U test; U = 477.5, P < 0.001). Epicephala moth eggs were laid at the basal part of the ovary, and oviposition occasionally damaged the ovule. Moth oviposition cut through the calyx lobes and the ovary wall, thereby leaving oviposition scars. These scars were reliable indicators of moth oviposition; the presence of scars and moth eggs had a one-to-one correspondence (N = 180). An egg-loaded flower had an average of 1.28 eggs (N = 141, range: 1–3).



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  		Fig. 17. Frequency distributions of the number of pollen grains attached to stigmas of female flowers (upper) and number of intact seeds per fruit (lower) in Breynia fruticosa at Dongmakhai. The mean number of pollen grains attached to female flowers with and without moth eggs was 14.58 ± 1.00 grains (mean ± SE; N = 141) and 1.07 ± 0.37 grains (N = 69), respectively

 
Seed destruction was caused exclusively by Epicephala moths, and a single larva destroyed all six seeds of a developing fruit (Fig. 17). However, 66.0% of the fruits remained uninfested (N = 43; Fig. 17). Of these intact fruits, 51.6% had oviposition scars, indicating that the eggs or early instar larvae of the moth did not develop in these fruits.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Obligate pollination mutualism in Breynia
This study revealed that at least two species of Breynia are pollinated by Epicephala moths that actively transport pollen between flowers. Although we did not observe moth visits in B. fruticosa, the presence of moth eggs in the majority of pollinated female flowers (Fig. 17), combined with possession of heavy pollen load on the proboscises of the female moths (Fig. 14), strongly indicates that Epicephala moths associated with B. fruticosa are the pollinators of their hosts. In both B. vitis-idea and B. fruticosa, larvae of the moths consumed developing seeds, but in total, a fraction of the seed crop was left intact, thus imposing a net benefit to plant reproduction (Table 2; Fig. 17).

Although our data showed that pollinated flowers of the two species normally had moth eggs (Figs. 15, 17), a fraction of fruits was not infested by Epicephala moths (Table 2; Fig. 17). In most cases, this is brought about by egg/larval mortality of Epicephala moths, as inferred from the indications of moth oviposition (egg remains in B. vitis-idea and oviposition scars in B. fruticosa) in these fruits. Yet, some fruits seemed not to have been oviposited by Epicephala (Table 2; Fig. 17). In B. vitis-idea, the proportion of intact fruits with moth oviposition was likely underestimated because egg remains are not always detectable and may be lost in the course of fruit development. Therefore, the possibility that the fruits normally had been oviposited cannot be ruled out in B. vitis-idea. In B. fruticosa, on the other hand, scars on calyx lobes and ovary walls are reliable markers of moth oviposition; thus, a fraction of intact fruits probably had not been oviposited. Pollination in these fruits may have been caused abiotically or by other potential copollinators such as gall midges. It is also possible that these fruits were produced by Epicephala moths that pollinated flowers but failed to lay eggs, as suggested for the Gomphidium–Epicephala mutualism (Kawakita and Kato, 2004 ). In any case, a detailed study of moth oviposition and the demographic pattern of moth egg/larvae are needed to fully understand the factors limiting the cost and benefit of the mutualism in these systems.

In some yucca–yucca moth interactions, yuccas selectively abscise flowers with high egg loads, thereby limiting seed destruction by yucca moths and allocating available resources to increased seed production (Pellmyr and Huth, 1994 ; Ritcher and Weis, 1995 ; Wilson and Addicott, 1998 ; Addicott and Bao, 1999 ). However, such a mechanism is not likely in the systems we studied, nor in the Gomphidium–Epicephala system (Kawakita and Kato, 2004 ), because the plants did not abort flowers with the level of egg load that most likely led to total destruction of seeds (two and one eggs in B. vitis-idea and B. fruticosa, respectively). Instead, egg/larval mortality of Epicephala moths played an important role in limiting seed consumption in the two systems. The importance of egg/larval mortality as a factor limiting excessive consumption is also noticed in other seed-parasitic pollination mutualisms including yucca–yucca moths (Addicott and Bao, 1999 ; Shapiro and Addicott, 2003 ), globeflower–globeflower flies (Jaeger et al., 2001 ), and senita cactus–senita moth mutualisms (Fleming and Holland, 1998 ; Holland and Fleming, 1999 ), as well as in an ecologically analogous fly–fungus mutualism (Bultman et al., 2000 ). Whether egg/larval mortality is host-induced (i.e., retaliation on overexploitation by the mutualist) or caused by factors independent of hosts is unknown. These issues should also be addressed in the future.

The genus Breynia currently comprises 35 species distributed in tropical regions of Asia, Australia, and the Pacific Islands (Govaerts et al., 2000 ). Plants of this genus are characterized by the fused, obconic or turbinate calyx lobes in male flowers and minute styles that are more or less fused in female flowers (Figs. 3, 4, 6, 7; Chakrabarty and Gangopadhyay, 1996 ). These structures likely prevent effective contact with anthers and stigmas by facultative flower visitors and suggest that the specialized Epicephala moth pollination is potentially widespread within the genus. Fruits of B. distica in New Caledonia and B. cernua and B. oblongifolia in Australia are also infested by Epicephala moths (A. Kawakita and M. Kato, personal observations), which further supports the widespread occurrence of obligate pollination mutualism in the genus Breynia.

Obligate pollination mutualism in Phyllanthaceae
Obligate pollination mutualism in Phyllanthaceae was first discovered between Glochidion trees and Epicephala moths (Kato et al., 2003 ). In this association, female Epicephala moths actively pollinate flowers and lay eggs in female flowers. The moth larvae consume the developing seeds, but on average, 20–54% of the seeds are left intact in each fruit (Kato et al., 2003 ). Species of Gomphidium are also pollinated actively by female Epicephala moths that oviposit in flowers (Kawakita and Kato, 2004 ). In this association, however, a single moth larva consumes the entire seeds of the developing fruit. Instead, 60–78% of the fruits are left untouched by the moth, probably because egg/larval mortality is high in these species or the moths do not always oviposit in flowers that they pollinate (Kawakita and Kato, 2004 ). The Breynia–Epicephala mutualism reported in this study is similar to the Gomphidium–Epicephala system in that the moth larvae frequently consume all seeds of the developing fruit (Figs. 16, 17). We showed that in this association, egg/larval mortality of Epicephala is an important, yet not exclusive, factor limiting seed destruction by the moths (Table 2, Fig. 17).

The different modes of plant–moth association found in Glochidion, Gomphidium, and Breynia provide multiple sources of variation in the factors affecting the interaction between Epicephala moths and their hosts. For example, ovule number per flower and fruit size typically vary among plant genera (e.g., six ovules per flower in Gomphidium and Breynia and 6–12 in Glochidion), which may be associated with the proportion of seeds that a single moth larva destroys. Also, oviposition methods vary among Epicephala moths infesting different host species (e.g., whether oviposition penetrates the ovary or not), and these differences may correspond to differential abilities of the host plants in detecting moth oviposition and selectively aborting heavily infested fruits, as suggested for some yucca species (Addicott and Bao, 1999 ; Marr and Pellmyr, 2003 ; Shapiro and Addicott, 2003 ). Thus, these variations allow comparative approaches in various ecological and evolutionary studies of plant–insect mutualisms. Furthermore, the interaction between the plant of a closely related genus Flueggea and its seed-parasitic Epicephala moth likely represent a plesiomorphic, antagonistic condition for the mutualism (A. Kawakita and M. Kato, unpublished data). Together, these attributes of the association between Phyllanthaceae plants and Epicephala moths provide an increasingly fascinating model system for studies of mutualisms and coevolutionary processes.


   FOOTNOTES
 
1 The authors thank Y. Kosaka and P. Thavy for invaluable help throughout their stay in Laos; S. Takeda, C. Houngphet, and the staff of National University of Laos for their kind support during field studies in Laos. This study was supported by the Japan Ministry of Education, Culture, Science, Sport, and Technology Grant-in-Aid for Scientific Research (#12440217 and #15370012) and by the Research Institute for Humanity and Nature and their grant for Research on Human Activities in Northeastern Asia and the Impact to the Biological Productivity in Northern Pacific Ocean. Back

2 kawakita{at}s01.mbox.media.kyoto-u.ac.jp Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Addicott J. F. 1986 Variation in the costs and benefits of mutualism: the interaction between yuccas and yucca moths. Oecologia 70: 486-494[CrossRef][Web of Science]

Addicott J. F. T. Bao 1999 Limiting the cost of mutualism: multiple modes of interaction between yuccas and yucca moths. Proceedings of the Royal Society, London, B 266: 197-202[Abstract/Free Full Text]

APG [Angiosperm Phylogeny Group]. 2003 An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG II. Botanical Journal of the Linnean Society 141: 399-436[CrossRef]

Bultman T. A. M. Welch R. A. Boning T. I. Bowdish 2000 The cost of mutualism in a fly–fungus interaction. Oecologia 124: 85-90[CrossRef][Web of Science]

Chakrabarty T. M. Gangopadhyay 1996 The genus Breynia (Euphorbiaceae) in the Indian subcontinent. Journal of Economic and Taxonomic Botany 20: 501-512

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