Beyond pollination: Ants and camel crickets as double mutualists in a non‐photosynthetic plant

Pollination and seed dispersal are essential processes in the plant life cycle, playing a crucial role in maintaining ecosystem functionality. These processes largely depend on animals (Ollerton et al., 2011). Most plants that are pollinated and dispersed by animals rely on different taxonomic groups for each function. However, there are instances of double mutualism where certain animal species provide both pollination and seed-dispersal services to the same plant species (Hansen & Müller, 2009). While double mutualism has been primarily perceived as anecdotal, a recent review identified approximately 300 potential cases (Fuster et al., 2019). Interestingly, almost all identified double mutualisms involve vertebrates (birds, mammals, or reptiles) (García et al., 2012). The sole robust exception is the interaction between the herb Borderea chouardii and ant species, with the genus Lasius facilitating pollination and the genus Pheidole facilitating seed dispersal (García et al., 2012). Recent research suggests that the transition to complete heterotrophy in plants often involves multiple phenotypic changes (Roy et al., 2013). A common characteristic among partially and fully heterotrophic plants is the significant reduction in seed size and complexity (Eriksson & Kainulainen, 2011). For instance, Balanophora fruits are among the smallest, with thousands of tiny dry fruits located at the base of a fleshy, club-shaped structure (the claviform body), which is a modified bract (Hansen, 1972). Although wind was considered the dominant dispersal mechanism in Balanophora (Ridley, 1906), the low wind speeds in dense forest habitats and the ovoid or cylindrical shape of their fruits likely limit the effectiveness of wind dispersal. Recent studies have shown that both birds and the Amami rabbit are key vectors for seed dispersal in Balanophora species with bright scarlet infructescences (Suetsugu, 2020a; Suetsugu & Hashiwaki, 2023). However, some Balanophora species, such as B. subcupularis, which have dull coloration and a yeasty odor (Figure 1A,B), may use different seed dispersal methods. Intriguingly, we have recently revealed that B. subcupularis is pollinated by ants, camel crickets, and cockroaches (Figure 1C,D). Moreover, our preliminary observations revealed that the camel cricket Diestrammena robusta feeds on the mature infructescences of B. subcupularis (Figure 1E). Although seed dispersal via insect excretion has rarely been recognized (de Vega et al., 2011; Duthie et al., 2006; Suetsugu, 2020b), our observations suggest a potential role for D. robusta in this process. This might represent a novel double-mutualistic relationship, especially considering our simultaneous identification of the camel cricket as a pollinator of B. subcupularis (Suetsugu & Hashiwaki, 2024a). We conducted extensive field observations and laboratory experiments to investigate the seed dispersal mechanism of B. subcupularis, focusing on the possible involvement of D. robusta. This research was carried out on Shimoshima Island, Amakusa Islands, in Kumamoto Prefecture, southwestern Japan. The details of field observation and laboratory analysis are provided in Appendix S1: Section S1. Our direct observations and time-lapse photography demonstrate that the camel cricket D. robusta and the ground beetle Synuchus spp. primarily forage on the fruits and claviform bodies of B. subcupularis (Table 1). In addition, the ant species Aphaenogaster famelica and Pheidole nodus regularly transport the fruits of B. subcupularis along with their claviform bodies (Figure 1E,F). However, Crematogaster osakensis, another ant species frequently visiting B. subcupularis infructescences, rarely carries the fruits due to its small mandibles. On the other hand, our infrared-triggered camera traps did not capture any mammals or birds feeding on the infructescences of B. subcupularis. Additionally, our field observations did not find any indications of bird or mammal feeding on the infructescences, ruling out seed dispersal by these groups. Ants are known to effectively disperse the seeds of at least 11,000 plant species, with this system evolving independently in over 100 lineages (Lengyel et al., 2009). A key adaptation in this process is the production of an elaiosome, a small food body that attracts ants (Lengyel et al., 2009). Interestingly, Balanophora seeds, despite lacking elaiosomes, are still dispersed by ants. It is possible that the claviform bodies, often carried along with the fruits, serve as edible rewards that encourage seed dispersal. Although some ants act as seed predators, we observed that A. famelica and P. nodus occasionally dropped the fruits or discarded them in refuse piles, thus facilitating seed dispersal. Despite ants generally being regarded as poor pollinators (Beattie et al., 1985), our concurrent study indicates they contribute to the pollination of B. subcupularis (Suetsugu & Hashiwaki, 2024a). Thus, the present findings, coupled with our complementary study, provide strong evidence of rare double mutualism between plants and ants. Our study also reveals that the camel cricket D. robusta plays a role in the seed dispersal of B. subcupularis. Feeding experiments and field collections showed that D. robusta defecated many seeds that were morphologically intact but had reduced survival rates (from 51.1% ± 13.0% [mean ± SE, number of mature infructescences from which intact fruit was collected: n = 5] to 10.6% ± 3.8% [n = 7: number of D. robusta individuals examined]) (p < 0.05; Figure 1G–I). Nevertheless, the decrease in seed viability does not entirely negate the potential for mutualistic relationships. Although small mammals predominantly consume fruits, often destroying the seeds, they can still act as mutualists by defecating some viable seeds (Suetsugu & Hashiwaki, 2023). Additionally, the seed dispersal of the host-specific obligate parasite B. subcupularis likely requires placement near appropriate host roots, which may be facilitated by camel crickets. We observed that they often inhabit crevices formed by the roots of Toxicodendron succedaneum, the compatible host of B. subcupularis. In contrast, the ground beetle Synuchus spp., a known seed predator, did not defecate intact seeds. It appears the seed testa offers partial protection in the camel cricket digestive tract but is not robust enough to withstand the mastication by Synuchus spp., whose mandibles are adapted to crush hard seed coats. Insects are typically not regarded as effective agents for endozoochorous (internal) seed dispersal, but an exception exists in the form of the weta, a large, flightless orthopteran in New Zealand. Wetas occupy ecological niches similar to small mammals, which are largely absent in New Zealand, excluding bats (Duthie et al., 2006). However, endozoochory can occur in any situation where seeds are ingested whole and are resilient enough to survive gut passage (Suetsugu, 2018, 2020b). This suggests that many small-seeded plants might use insect-based dispersal systems, even in environments with small mammals. Indeed, camel crickets have been identified as seed dispersers for several heterotrophic plants in Japan (Suetsugu, 2018, 2020b). Importantly, our findings represent the first report of invertebrate-mediated seed dispersal in the Balanophoraceae. We propose that claviform bodies may play a vital role in seed dispersal across different Balanophora species, as indicated by their complete digestion by D. robusta and consistent transportation by ants. This aligns with findings that claviform bodies in other Balanophora species act as rewards for birds and the Amami rabbit (Suetsugu, 2020a; Suetsugu & Hashiwaki, 2023). Although the ecological function and evolutionary origins of the claviform body have remained unclear (Eberwein et al., 2009), our observations indicate that it may have evolved for seed dispersal, providing insight into the life history of Balanophora species. In summary, our discovery that camel crickets and ants fulfill dual roles as pollinators and seed dispersers represents a rare instance of double mutualism facilitated by invertebrates. Notably, this study underscores the role of camel crickets as dual agents of pollination and seed dispersal, marking the first known case of double mutualism involving invertebrate species acting as endozoochorous seed dispersers. Double mutualism is more common in isolated environments such as islands, where plants rely on a few potential partners (Fuster et al., 2019). The unique conditions of our study site, such as a small island hosting the northernmost population of B. subcupularis, might contribute to the scarcity of pollinators and seed dispersers, leading to this novel double-mutualistic interaction. Future research is necessary to assess the extent of such mutualisms across the distribution range of B. subcupularis. We express our gratitude to Masato Watanabe and Hiroyuki Iwamoto for their invaluable support during the field studies. We also thank Akihiko Ichikawa, Akira Yamawo, and Toshio Kishimoto for their assistance with insect identification. The authors also thank Kazuma Takizawa, Hidehito Okada, and Suzu Kawashima for their assistance in the laboratory work. This study was financially supported by PRESTO (JPMJPR21D6, Kenji Suetsugu) from the Japan Science and Technology Agency. This study received financial support from the JST PRESTO grant number: JPMJPR21D6. The authors declare no conflicts of interest. Data (Suetsugu & Hashiwaki, 2024b) are available in Figshare at https://doi.org/10.6084/m9.figshare.25465483. Appendix S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.