Ant-plant mutualisms should be viewed as symbiotic communities

Abstract

Ant-plants provide food and nesting space (domatia) for ants that protect them against herbivores. These mutualisms are often very specific and are usually considered as bipartite, or tripartite when ants use hemipterans as trophobionts. However, fungi growing inside domatia have been recorded by a few authors. Here we report on their occurrence on additional ant-plants from Africa, Asia and South America. We demonstrated the symbiotic nature of the relationship between the plant, the ant and the fungus in the model plant Leonardoxa africana africana and its mutualistic ant Petalomyrmex phylax. Moreover, data suggest the ant-fungus relationship is mutualistic. Here we discuss the most probable role of the fungus and the potential implications on the understanding of nutritional ecology of ant-plant symbioses. The fungus is also associated with the presence of nematodes and bacteria. Many ant-plant symbioses previously considered to be bipartite will soon likely prove to be multipartite symbiotic communities.

In tropical forests, some plants known as ant-plants, or myrmecophytes, are engaged with ants in tight, often obligatory relationships. These plants have specialised hollow structures, called domatia, to host ant colonies. Such structures involve stems, thorns, stipules, petioles or leaves, depending on the species. Moreover, these plants produce extrafloral nectar and/or food bodies to sustain their ant colony. In mutualistic interactions, ants contribute to protecting the plant against herbivores,¹ fungal pathogens² and competing plants.³ These mutualisms have focused the attention for decades because of the spectacular nature of coevolved traits. At the beginning of the 20^th century, a few authors reported the presence of fungi within ant-plants belonging to a total of 11 genera.^4–7 Within the last four decades, publications on ant-plants have increased at an exponential rate, but very few authors have taken fungi into account.^8,9 Recently, we described new cases of fungi growing in ant-plants and, for the first time, investigated the nature of the relationship.¹⁰

The ant-plant Leonardoxa africana africana (Fabaceae) is endemic to a narrow strip of coastal lowland rain forest in southern Cameroon.¹¹ Each plant is occupied by a single colony of the mutualistic ant Petalomyrmex phylax.¹² However, the mutualistic colony is sometimes replaced by Cataulacus mckeyi, an ant considered as a parasite of the mutualism because it takes advantage of nesting space and foliar nectar without protecting the plant.¹³ We now have dissected more than 1,200 domatia from more than 150 plants occupied by P. phylax. More than 95% of the domatia with the mutualistic ants have fungus. On the other hand, unopened domatia and domatia from unoccupied plants or plants occupied by C. mckeyi bear no fungus. Thus, the occurrence of the fungus is strictly tied to the presence of the mutualistic ant. Moreover, in young domatia that have been only very recently opened and occupied by the ants, the fungal patch is not distinguishable by visual inspection, but a pile of very small pieces of “rubble” or detritus is present. As domatia become older a very small patch of fungus is seen to be associated with the pile of “rubble”. Thus, mutualistic ants may actively propagate the fungus. Recording ant behaviour inside the domatia with an endoscope revealed another specific ant behaviour: on a few occasions ants were seen chewing the fungal mat and cutting off a piece of it. This could explain the regular and highly localized size of fungal patches, a very unusual feature to any mycologist's eye.

Sequencing internal transcribed spacer (ITS) of ribosomal DNA of the fungi from four individuals of L. a. africana showed the same species across samples. In this system, the plant-ant-fungus association seems specific. This fungus, as well as those identified from two other ant-plants (Barteria fistulosa [Passifloraceae] and Keetia hispida [Rubiaceae]), belong to the Ascomycota order Chaetothyriales. Ant-mediated propagation of the fungus, specific ant behaviour toward the fungus and species specificity of the association clearly demonstrate the symbiotic nature of this plant-ant-fungus association. All the P. phylax colonies we investigated (more than 150, all over the distribution area) had fungus in their host plant. The ant-fungus association is thus constant. Moreover, domatia that we disturbed in the course of our experiments (by repeatedly drilling and manipulating them) were eventually deserted by the ants and the fungus, suggesting that the ants had removed it when they fled. Such behaviour toward a parasite is rather unlikely. Thus, data from our study model suggest that the ant and the fungus have rather a mutualistic relationship. But still, the mutualistic nature of the relationship remains to be demonstrated. Other ant-plant systems have to be investigated to fully understand evolutionary diversification of these ant-plant-fungal symbioses.

Whether the fungus is mutualistic can be established by elucidating its role in the threeway system. Fungi are used by the ants Allomerus decemarticulatus and Lasius, respectively to build a trap facilitating the capture of large prey¹⁴ and to reinforce nest walls (occasionally involving Chaetothyriales).¹⁵ In each domatium of L. a. africana, the fungus appears in the form of a thin discoid patch, usually less than 1 cm in diameter, of hyphae. A structural role of the fungus is thus very unlikely. In fact, only two hypotheses appear plausible. First, the fungal patch could serve as a nutrient recycler, allowing all the refuse produced by the colony to be transformed into nutrient forms available to the plant, or even to the ants. Second, the fungus could be a food source for the ants, as is the case for Attine ants¹⁶ and for Euprenolepis procera, which relies entirely on naturally growing mushrooms.¹⁷ These two hypotheses are not mutually exclusive. Indeed, almost every fungal patch is associated with the refuse pile of the colony containing fragments of insect cuticle in which we recognised parts of the mutualist ants, among other unidentifiable debris. By contrast, domatia occupied by the parasitic ant C. mckeyi, which do not have fungus, do not contain a refuse pile. Moreover, ants were seen defecating on the fungal patch. These observations argue in favour of the first hypothesis. But the chewing of the fungus in a stereotyped manner is reminiscent of pruning behaviour by the New World fungus-growing ants (Attini), which grow fungus as a staple food.

Nutrient balance in plant-ants is considered nitrogen-limited because plant exudates and honeydew constitute supposedly the most important part of their diet.^18–20 Symbiotic bacteria have been identified in the gut of several ant species.^21,22 Such symbionts in plant-ants could provide essential amino acids or help fixing nitrogen. If the fungi grown by plant-ants prove to be a food source, it would certainly revolutionize our understanding of the nutritional ecology of ant-plant symbioses.

Our early stage investigation of ant-plant/ant/fungi relationships led us to realise that more organisms may be involved in domatia functioning. First, nematodes abund in fungal patches of almost all species. They were already noticed in some of the early descriptions of the fungi. They can be seen under the binocular and their movements can even be detected by the naked eye, especially in the large fungal patches in B. fistulosa occupied by T. aethiops. Those we identified from colonies of the plant-ants T. aethiops and P. phylax belong to the genus Pelodera, nematode grazers feeding on bacteria.²³ Second, transmission electron microscopy on the fungal patches revealed a world of microorganisms including numerous bacteria. Moreover, many plant-ants are known to rear hemipterans for food within domatia.²⁴ The presence of hemipterans and fungi is not exclusive because several plant-ants, such as Aphomomyrmex afer (in L. a. letouzeyi), T. aethiops (in B. fistulosa) and Crematogaster (in K. hispida), have both. Even if the roles of the newly identified partners in ant-plant symbioses are not yet fully understood, the potential for discovering new symbionts is great. It is noteworthy that the first organisms to be identified in the ant-plant symbioses were the plant, the ant and the hemipterans, all three big enough to be obvious to the naked eye. Then, the less evident fungi and nematodes came on stage. And the invisible microorganisms will probably do so in the near future. This discovery sequence is certainly due to technological limitations and raising interest for the invisible biodiversity. It is highly reminiscent of the story of fungus-growing ant research. Despite the ecological and economic importance of some attines (Atta is a pest of agriculture in the neotropics), their symbiotic relationship with fungi was evidenced only at the end of 19^th century. Attine-fungus symbiosis started to be considered as a symbiotic community only 20 years ago, when ants were discovered to host specialised antibiotic-producing bacteria to protect their mutualistic fungus against a fungal parasite.^25,26 Only recent advances in molecular technology allow the identification of new symbionts and elucidation of their roles.²⁷ We are thus confident that similar tools applied to ant-plant associated communities will reveal a complex symbiotic community.

In addition to the 11 ant-plant genera reported to host fungi at the beginning of the 20^th century, our own investigations (including very recent findings) showed fungi in six more genera: Leonardoxa, Calpocalyx (Fabaceae), Keetia (Rubiaceae) and Vitex (Lamiaceae) from Cameroon,¹⁰ Tachigali (Fabaceae) from French Guiana, and Drypetes (Euphorbiaceae) from Borneo (kindly provided by Joachim Moog). The preliminary dissection of a few leaf pouches of Tococa (Melastomataceae) and hollow stems of Cecropia (Cecropiaceae) from French Guiana did not show any visible fungal patch. Nevertheless, fungi occur in tropical ant- plants symbioses on all continents. This is thus a very common and widespread phenomenon, which has been overlooked. The small window opened on these symbiotic communities has highlighted many promising research directions.

Addendum to: Defossez E, Selosse MA, Dubois MP, Mondolot L, Faccio A, Djieto-Lordon C, et al. Ant-plants and fungi: a new threeway symbiosis. New Phytol. 2009 doi: 10.1111/j.1469-8137.2009.02793.x. In press.