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A novel brood-site pollination mutualism?: the rootholoparasite Thonningia sanguinea (Balanophoraceae) andan inflorescence-feeding fly in the tropical rainforests ofWest Africapsbi_338 164..169

RYUTARO GOTO,* GEN YAMAKOSHI† and TETSURO MATSUZAWA‡*Graduate School of Human and Environmental Studies, Kyoto University, Yoshida-Nihonmatsu-cho, Sakyo, Kyoto 606-8501,Japan, †Graduate School of Asian and African Area Studies, Kyoto University, 46 Shimo-Adachi-cho, Sakyo, Kyoto 606-8501,Japan and ‡Primate Research Institute, Kyoto University, Inuyama, Aichi 484-8506, Japan

Abstract

The Balanophoraceae is a unique angiosperm family that fully parasitizes the roots oftrees. Although the pollination systems of several genera in this family have beenreported, little is known of their diversity. In the present study, we investigated thepollination biology of Thonningia sanguinea (Balanophoraceae) in the tropical rainforestsof Guinea, West Africa. Female flies of the families Muscidae and Calliphoridae as wellas Technomyrmex ants frequently visited flowers to consume nectar secreted from inflo-rescences. While feeding, their bodies attached to anthers or pollen grains. The mostabundant flower-visiting fly, Morellia sp. (Muscidae), was observed laying eggs on T. san-guinea, and the larvae fed only on the vegetative tissue of decaying male inflorescences.Our findings provide a new candidate of pollination mutualism involving plants thatprovide brood sites for their pollinators.

Keywords: Balanophoraceae, brood-site pollination, male-plant-biased herbivory, Morellia,Thonningia.

Received 14 December 2010; revision received 1 April 2011; accepted 11 April 2011

Introduction

Pollination mutualisms involving plants that offer pollen,nectar or both to pollinating insects as a reward for pol-lination are common in angiosperms (Protocor et al.1996). However, pollination mutualisms involving plantsthat offer a brood site to insects as the pollination rewardare very rare (Sakai 2002). Nevertheless, these mutual-isms are often treated as model systems of coevolutionand mutualism because they often exhibit highly intimateinteractions or extreme co-specialization between plantsand pollinating insects (Thompson 1994, 2005). The obli-gate mutualisms of the fig–fig wasp and yucca–yuccamoth systems, whereby the plants offer seeds or ovulesas brood sites for pollinators, are the best-knownexamples (Janzen 1979; Pellmyr 2003). Over the past

three decades, various brood-site pollination systemshave been discovered in various plant groups(Silberbauer-Gottsberger 1990; Sakai 2002; Kato et al.2003; Ishida et al. 2009; Kawakita 2010; Luo et al. 2010).Nevertheless, little is known about the diversity of thisunusual pollination system among the great diversity ofangiosperms. In the present study, we provide a newbrood-site pollination system candidate from the AfricanBalanophoraceae.

The Balanophoraceae is a subtropical to tropical familyof obligate parasitic angiosperms, consisting of 18 generaand approximately 50 species (Shumei & Murata 2003).The family is characterized by unique fungus-like inflo-rescences, with numerous tiny unisexual flowers (Holza-pfel 2001; Kawakita & Kato 2002). All members of thisfamily are entirely non-photosynthetic and parasitize theroots of host trees, depending exclusively on nutrientsexploited from the host. Although detailed information islimited to several genera, previous studies suggest that

Correspondence: Ryutaro GotoEmail: [email protected]

Plant Species Biology (2012) 27, 164–169 doi: 10.1111/j.1442-1984.2011.00338.x

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© 2011 The AuthorsJournal compilation © 2011 The Society for the Study of Species Biology

the pollination system is diverse in this family. In Balano-phora, an Indian species is pollinated by small honeybees(Govindappa & Shivamurthy 1975), whereas Japanesespecies are pollinated by ants and pyralid moths (Kawa-kita & Kato 2002). The two genera of pyralid moths usethe nutritive tissue of the flowers as brood sites (Kawakita& Kato 2002). In contrast, some Costa Rican species (e.g.Helosis and Corynaea) are pollinated by tachinid flies(Gomez 1983), a New Zealand species, Dactylanthus tay-lorii, is pollinated by short-tailed bats (Ecroyd 1996), andan Amazonian species, Lophophytum mirabile, is pollinatedby small beetles (Chrysomelidae, Nitidulidae, Staphylin-idae and Curculionidae; Borchsenius & Olesen 1990). Thelatter species is believed to offer the beetles nutritivetissue for brood sites or mating sites, although detailedobservations are lacking. Interestingly, despite a paucity ofpollination studies, two different brood-site pollinationsystems have been discovered in this family (Borchsenius& Olesen 1990; Kawakita & Kato 2002). Certain morpho-logical characters of Balanophoraceae, such as the fleshyaxis of the inflorescence, may perhaps favor the evolutionof brood-site pollination mutualism. To further clarify theecological conditions under which brood-site pollinationevolved and the reproduction biology of this unusualparasitic plant family, investigations of more genera inBalanophoraceae are required.

Thonningia, a dioecious genus of Balanophoraceae, isendemic to the tropical rainforests of Africa (Hutchinson& Dalziel 1958; Letouzey 1986). This genus includes onlyone variable species, Thonningia sanguinea, which parasit-izes the roots of a wide range of host trees (Olanya & Eilu2009). In the present study, we investigated the pollinationsystem of T. sanguinea and the biology of the muscid fliesthat use T. sanguinea as a brood site.

Materials and methods

Study species and study sites

The study site is located in the tropical rainforest of theGban and Guein Hills, near Bossou village in southeast-ern Guinea, West Africa (7°39′N, 8°30′W; 500–700 m a.s.l.).Traditionally the forests of this area have been protectedby the local village people as part of their religion (Yama-koshi 2011). The local temperature averages 20–30°C andrainfall is 2000–2500 mm per year (Yamakoshi 1998; Take-moto 2004). The climate is characterized by distinct rainy(March–October) and dry (November–February) seasonsYamakoshi 1998). This site is well known for long-termfield studies of wild chimpanzees that began in 1976 (Sug-iyama 1981; Matsuzawa et al. 2011).

Thonningia sanguinea is a low-growing perennial plantthat parasitizes the roots of a wide range of host trees(Fig. 1; Olanya & Eilu 2009). Plants are either male or

female (dioecious) and produce inflorescences at approxi-mately 4 cm above ground level (Fig. 1a–d,g). Inflores-cence tops are densely covered with numerous minuteflowers. Both male and female flowers essentially lackperianths (Hutchinson & Dalziel 1958), and the inflores-cence is covered by protruded anthers in male plants andstyles in female plants, giving overall white and yellowappearances, respectively, to the flowers (Fig. 1a–d,g). Theplants are surrounded by thick, red, scaly leaves (Fig. 1),and the infructescence is reddish, spherical and coveredwith numerous seeds (Fig. 1h).

Field observations, collection of flower visitors andinsect rearing

Observations were basically limited to male inflorescencesbecause of the very low density of female inflorescences inAugust 2005. On the gloomy forest floor in the GueinHills, we observed two patches of T. sanguinea, one com-posed of a single male inflorescence and one inflorescencebud (Fig. 1a) and the other comprising one female inflo-rescence. Flower visitors to the male inflorescences wereobserved for 11 h (07.00–18.00 hours) on 31 August 2005and those to the female inflorescence for 0.5 h (12.00–12.30 hours) on 1 September 2005. We recorded flower-visiting insects and collected each species foridentification, mainly in the late afternoon. Insect visits tothe flowers were counted when any body part of thevisiting insect contacted the anther. We also recordedinsects visiting the inflorescence bud that adjoined themale inflorescence (Fig. 1a). After field observations, driedspecimens of the collected flower visitors were observedunder a microscope to check for pollen attachment to theirbodies.

To investigate insect feeding on T. sanguinea, nine maleinflorescences, one female inflorescence and eight fruitswere collected from six patches of T. sanguinea. Soon aftercollection, we dissected a subset of the samples andlooked for insects feeding on them. If such insects werefound, we recorded the number and stored them in 100%ethanol for subsequent identification. The remaininginflorescence and fruit samples were kept at local tem-perature in separate plastic bags to obtain adult insects.

Results

Observations of flower visitors

During the 11-h observation period of a male inflores-cence, flies made 66 floral visits, and massive ant floralvisits were observed several times (Figs 1a–d and 2a).Both flies and ants fed on nectar secreted from the inflo-rescences (Fig. 1a–d). While feeding, their bodies fre-quently contacted an anther (Fig. 1a–d). Floral visits by

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Fig. 1 Thonningia sanguinea and its pollinators. (a) One male inflorescence and one bud of T. sanguinea; (b) Morellia sp. (Muscidae) visitinga male inflorescence of T. sanguinea; (c) Calliphora sp. 1 (Muscidae) visiting a male inflorescence of T. sanguinea; (d) Technomyrmex sp.(Formicidae) visiting a male inflorescence of T. sanguinea; (e) Morellia sp. ovipositing onto a red scaly leaf of T. sanguinea; (f) larvae feedingon the fleshy axis of a decomposing male inflorescence; (g) a female inflorescence of T. sanguinea; the eggs of Morellia sp. are attached tothe red scaly leaves; (h) a mature infructescence of T. sanguinea.

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flies continued during the observation period, but visitfrequency was low around noon (Fig. 2a). In contrast,floral visits by ants were high at that time (Fig. 2a).Flower-visiting flies comprised one species of Muscidae(Morellia sp.) and two species of Calliphoridae (Calliphorasp. 1 and Calliphora sp. 2). All of the collected specimenswere female (Table 1). Flower-visiting ants were all asingle Technomyrmex species. Pollen grains were mainlyattached to the legs of flies and to the mouthparts of ants(Table 1). Unlike other flower-visiting flies, the most fre-quent floral visitor, Morellia sp., also frequently visited aninflorescence bud next to the flowering male plant underobservation (Fig. 2b). These flies oviposited onto the scaly

leaves twice during the observation period (Figs 1e, 2b,arrows), laying two or three eggs on the leaves per visit. Inall, nine and seven eggs were observed on the male inflo-rescence and inflorescence bud, respectively.

We could not adequately observe floral visits by polli-nators to the female plant because of the short observationtime (0.5 h). However, female inflorescences also secretenectar from nectarines similar to those of male inflores-cences. Furthermore, we found 12 Morellia sp. eggs on thescaly leaves of the female plant (Fig. 1g), suggesting fre-quent visits by Morellia sp. to female plants.

Observations on larval feeding biology

We collected nine male inflorescences, one female inflo-rescence and eight mature fruits from six patches of T. san-guinea. Fly larvae were observed on all of the maleinflorescences, but not on any female inflorescences orfruits. We carefully dissected five decomposing maleinflorescences soon after collection and found fly larvaeand feeding traces of larvae that extended from the top tothe base of the decaying male inflorescences (Fig. 1f). Onaverage, male inflorescences contained 3 � 0.84 (1–5)larvae. Furthermore, seven larvae exited from the remain-ing four inflorescences kept in plastic bags. Feeding tracesof fly larvae were also found on all of these inflorescences.Larvae pupated soon after escaping from the inflores-cences. Approximately 30 days later, adult flies emergedfrom all of the pupae (n = 7) and were identified asMorellia sp.

We also dissected five mature fruits soon after collec-tion, but found no larvae or feeding traces. Although wekept one female inflorescence and three fruits for approxi-mately 2 weeks, no insects escaped from the mature fruits.Dissection of the inflorescence and fruits revealed nolarvae or feeding traces.

Discussion

In the present study, we found that a male inflorescence ofT. sanguinea was visited mainly by female flies of the fami-lies Muscidae and Calliphoridae, and by Technomyrmexants, which consume nectar secreted from the inflores-cences (Figs 1a–d, 2a). The most abundant flower-visiting

Fig. 2 Diurnal patterns of insect visits to a male inflorescence andbud of Thonningia sanguinea. (a) Visit frequency per male inflo-rescence per hour and (b) visit frequency per inflorescence budper hour. Black and white boxes indicate flies and ants visitingthe inflorescence or bud, respectively. Flower-visiting flies aredivided into the muscids (Morellia sp.; black circles) and calli-phorids (Calliphora sp. 1 and Calliphora sp. 2; white circles).

Table 1 List of insects collected on a maleinflorescence of Thonningia sanguinea, theirsex and pollen attachment to their bodies

Order Family Species M F Pollen attachment

Diptera Muscidae Morellia sp. 0 18 ++Diptera Calliphoridae Calliphora sp. 1 0 6 +Diptera Calliphoridae Calliphora sp. 2 0 6 +Hymenoptera Formicidae Technomyrmex sp. 0 10 +

F, female; M, male; +, 1–10 grains; ++, 10–100 grains.



fly, Morellia sp., oviposited onto the scaly leaves and thelarvae fed on decaying male inflorescences (Fig. 1e–f).Although our observation of female inflorescences waslimited, the nectar secreted from both male and femaleinflorescences suggests that flower visitors to femaleinflorescences are similar to those visiting male inflores-cences. The eggs attached to female plant leaves suggestfrequent visits of Morellia sp. to female plant patches(Fig. 1g). The visits, combined with the observation thatthese flies frequently carry pollen on their bodies, suggestthat Morellia sp. represents an effective pollinator ofT. sanguinea. In Balanophoraceae, two cases of pollinationmutualisms involving plants that provide brood sites fortheir pollinators have been reported: Lophophytum mirabileand beetles in an Amazonian forest (Borchsenius &Olesen 1990) and Balanophora species and pylarid mothsin Japan (Kawakita & Kato 2002). Thus, T. sanguinea and itsinflorescence-feeding muscid fly in West Africa mayprovide a third example of a brood-site pollination systemin this family. A distinguishing feature of Balanophora-ceae is that fleshy vegetative tissue (inflorescence axis)occupies a large portion of the inflorescence mass,whereas the flowers constitute only a thin layer on thesurface of the inflorescence. This morphological charactermay offer herbivores a stable resource to be used forbrood sites, consequently leading to the evolution of abrood-site pollination system.

In most brood-site pollination systems, pollination isachieved via pollinator body contact with anthers orstigmas as the pollinator oviposits on the flowers (Sakai2002). However, the body of the Morellia fly does notcome into contact with anthers or stigmas when it ovi-posits onto a scaly leaf (Fig. 1e). This raises the questionof whether the brood-site use of the plant by flies con-tributes to the reproduction of the plants. However, weobserved that Morellia flies frequently visited flowers ofT. sanguinea while searching for a brood site in T. san-guinea patches. In addition, all of the Morellia sp. col-lected on the flowers were females, suggesting that floralvisits by Morellia sp. occur while searching for broodsites. These observations suggest that the use of T. san-guinea as a brood site by flies promotes the attraction ofMorellia sp. to the flowers.

In brood-site pollination mutualisms between plantsand pollinators whose larvae consume seeds or ovules,such as the fig–fig wasp and yucca–yucca moth mutual-isms, a high density of pollinators may result in excessiveseed destruction by the larvae of the pollinator, thus ham-pering plant reproduction (Janzen 1979; Pellmyr & Huth1994; Goto et al. 2010). However, in our study system,Morellia flies fed only on the vegetative tissue of decom-posing male inflorescences (Fig. 1f), not on fresh anthersor female inflorescences or fruits that are indispensablefor plant reproduction. Thus, the cost of pollination by

Morellia sp. for the host plant is probably negligible.However, why does Morellia sp. larval herbivory occuronly on male inflorescences? A similar phenomenon hasbeen observed in other brood-site pollination systems inwhich plants offer decomposing flowers to pollinators asbrood sites (Sakai 2002). In Zamia, male inflorescencescontain more starch than those of females, as a resultpollinating beetles prefer male inflorescences (Norstog &Fawcett 1989). In Artocarpus, the larvae of pollinating gallmidges feed solely on a fungus that grows only in maleinflorescences (Sakai et al. 2000). In T. sanguinea, the post-flowering development of male and female inflorescencesdiffers greatly and may affect the parasitism of Morellia sp.Male inflorescences decay slowly after blooming, whereasfemale inflorescences develop into fruits. Thus, femaleinflorescences most likely contain more anti-herbivorechemicals, resulting in larval death at an earlier stage thanmale inflorescences, even after blooming.

In contrast, we observed Morellia eggs on both male andfemale inflorescences (Fig. 1), which is somewhat puzzlingas Morellia larvae survive only on male inflorescences andnot on female inflorescences or fruits, thus potentiallyselecting for females that can distinguish between maleand female inflorescences. Therefore, eggs attached tothese inflorescences may have been laid when the inflores-cences were still in the bud stage, when the flies had noclues to distinguish between males and females.

All of the flower-visiting flies were female (Table 1).However, we did not observe oviposition behavior of non-Morellia flies or larvae emerging from T. sanguinea tissues.Why is the sex of these flies female biased? One possibilityis that the timing of emergence of male and female fliesdiffers. Alternatively, T. sanguinea may emit a signal toattract pollinators (e.g. a floral scent) that is detected orfavored only by female flies. Some plants attract femaleflies by mimicking the brood site using floral scents (Stens-myr et al. 2002). An investigation of T. sanguinea floralscents may provide a better understanding of plant–flyinteractions in this unusual pollination system.

Acknowledgments

This work was financially supported by a Japan Society forthe Promotion of Science (JSPS) for Young Scientists grant21-1986 to R. Goto, and JSPS-HOPE (Primate Origins ofHuman Evolution) and MEXT (Ministry of Education,Culture, Sports, Science and Technology) grants16002001–20002001 to T. Matsuzawa. We are grateful tothe Direction Nationale de la Recherche Scientifique etTechnologique, Republic of Guinea and l’Institut deRecherche Environnementale de Bossou for permission tocarry out this research and for approving it. We thank A.Kabasawa, Y. Sugiyama and P. Cherif for fieldworksupport; T. Okamoto and Y. Nakase for help with insect

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identification; H. Kurahashi for providing information onknown African Calliphoriidae; and M. Kato, A. Kawakitaand K. Suetsugu for constructive comments on this study.

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