Social animals have evolved into highly developed societies through intimate cooperative and altruistic behaviours1. For instance, the development of ‘agriculture’, managing other organisms for stable supply of food resources, requires highly sophisticated cooperative behaviours2. Indeed, some non-human animals are engaged in agriculture, of which the best-known examples are fungus-growing ants (tribe Attini, subtribe Attina). While the origin of human agriculture was only ~10,000 years ago, fungus-growing ants can be traced back to 60 million years ago3.
Agricultural ants have diverse societies
Ant agriculture involves garden cultivation of fungi (order Agaricales) within their nests to nurture the queen and larvae with balanced symbiotic fungi containing high levels of nutrients4. Fungus-growing ants compose 17 genera containing ~250 species and maintain a wide array of social structures often studied with respect to various evolutionary theories4. In groups that have retained ancestral traits (10 genera, ~130 species), there are only 50–100 workers in a nest, with no physical differences among them5. Fungal gardens are restricted to small spaces, such as shallow soils, rotten wood, or underneath rocks6. The advanced group (5 genera, ~80 species) have colony sizes of several hundred to a thousand individuals, multiple fungal garden chambers, and two or three physically identical subcastes7, 8.
The leaf-cutting ants (genera Atta and Acromyrmex; ~40 species), have the largest colonies and most complex societies among ants, with millions of individuals and >10 subcastes, ranging from small (2 mm in length) to large workers (>15 mm in length)3, 4, 6. As they require large amounts of vegetal substrates to cultivate numerous mutual fungi, these ants play a critical role in the ecological succession of vegetation in Neotropical forests7, 8. When their vigorous agricultural activities are directed towards human society, they cause enormous economic and agricultural damage. For instance, Atta texana in Texas can destroy >5,000 ha·yr-1 of pine seedlings, with annual control and management costs reaching ~US$ 2.3 million9.
To maintain such a complex social structure and elaborate agricultural system, leaf-cutters must perform >30 time-sensitive tasks with the most appropriate members6, 7, 8, and our preliminary research in which we recorded high-frequency acoustic signals in Atta led us to hypothesize that ‘talking’ (i.e. acoustic communication) is an important factor used to control such social behaviours.
Social evolution is based on communication
Eusocial insects, including fungus-growing ants, are conventionally referred to as ‘superorganisms’10, and the study of communication within these species is expected to provide a major breakthrough in the theory of social evolution11.
Highly evolved social behaviours of insects are controlled by constant sharing of information through chemical12, 13, acoustic14, 15, visual 16, 17, and tactile18 stimuli (Table 1). In ants, chemical communication is generally understood to be a foundational feature of their social evolution and cooperative behaviours12, 13. Although visual, acoustic, and tactile communications are postulated as the next evolutionary steps, those have yet to be experimentally revealed11.
Barbero et al. (2009) published a revolutionary study on acoustic communication in ants19. It had been established that larvae of the lycaenid butterfly (genus Maculinea) used chemical mimicry to invade host ant (Myrmica) nests20; additionally, the larvae of Maculinea (Phengaris) rebeli were found to mimic queen ant sounds of Myrmica schencki and received protection from the workers once inside the nest. The study revealed that queens sounds induce cooperative behaviours, bringing attention to the importance of acoustic communication among ants. Further examples of acoustic communication research are well known in leaf-cutting (genus Atta)21 and fire ants (Solenopsis invicta)22. Others are also known to produce drumming sounds by striking their abdomens against the ground23, 24; however, no quantitative studies have been conducted on these functions.
To establish the importance of acoustic communication in the evolution of sociality in fungus-growing ants, we proposed the following two hypotheses: (1) A significant correlation exists between social evolutionary levels and sound-production frequency and types; and (2) acoustic as well as chemical communication in agriculture-practicing ants require a high level of cooperative behaviour. To test these hypotheses, we obtained detailed stridulatory sound recordings with each independent behaviour of seven genera (eight species) of fungus-growing ants using a high-resolution recording device that we developed. Phonetic analyses of the recorded sounds and structural analysis of the sound-producing organs were carried out via SEM, and the effects of chemical and acoustic stimuli on fungus-garden maintenance were examined through their experimental inhibition.
‘Chattier’ ants formed complex societies
Using a self-developed, high-resolution recording device, we recorded the acoustic signals of 3–10 workers and fragments of symbiotic fungi from their fungus garden for 15–30 min in attines and successfully obtained ant-derived stridulatory sounds for all species (Extended Data 1-3). The groups that retained ancestral traits produced acoustic signals <0.5 times per minute per individual (0.01 ± 0.002 in Myrmicocrypta, 0.07 ± 0.06 in Apterostigma auriculatum, 0.03 ± 0.03 in Ap. mayri, 0.26 ± 0.30 in Cyphomyrmex). In the moderately derived group, the average frequency was 6.37 ± 2.32 and 2.15 ± 2.24 times per minute per individual in Trachymyrmex and Sericomyrmex, respectively. The genera of the highly advanced group, Acromyrmex and Atta, were much ‘chattier’ (average frequency 19.15 ± 6.35 in Acromyrmex and 24.08 ± 24.21 in Atta) than those of the other groups. A Jonckheere–Terpstra test confirmed a significant positive correlation between the stridulatory sound frequency and estimated branch age of each lineage (Z-value, 2.97; P < 0.01).
We next recorded and phonetically analysed acoustic signals under independent stimuli and external conditions for the ‘chattiest’ leaf-cutting ant species, Atta colombica. The two independent external stimuli were (1) pinching with forceps and (2) burying with oats, whereas the recorded signals from independent conditions and locations were (3) cutting Leguminosae leaves, (4) cutting Clusiaceae leaves, (5) cutting Heliconiaceae flowers, (6) vigilant sound near a fungus garden, (7) bark-like sounds near a garbage dump, (8) bark-like sound near an entrance, (9) alarm sound on a trail, (10) larval care, and (11) a queen’s alarm sounds were extracted and subjected to phoneme and canonical discriminant analyses. Each acoustic signal was discriminated at a rate of 60–100% (Fig. 1; Function 1: χ2 = 750.225; P < 0.0001, raw data: Extended Data 4, Supplementary Information). The variables that correlated best with axes 1 and 2 from the standardized discrimination coefficients were f0 duration (seconds) and pitch range, respectively. A 100% discrimination was found for four stimuli: (1), (3), (9), and (11). Although 14 types of stridulatory sounds were significantly discriminated in preliminary analysis, including those of workers of different body sizes (2.5–15.0 mm in body length), only the acoustic signals produced by medium-sized workers (~7.0 mm) and queens were recorded here; thus, the effects of the size of the stridulatory sound-producing organs due to differences in body size were eliminated, and only differences between situations, stimuli, and acoustic signal types were observed. Reanalysis of seven types of mixed acoustic signals (Fig. 1a, dashed circle) resulted in discrimination rates of 80–100% (Fig. 1b). A 100% discrimination was revealed for (2), (4), (5), and (7) (Function 1: χ2 = 453.974; P < 0.0001). From the standardized discrimination coefficients, the strongest correlations for axes 1 and 2 were f0 start (Hz) and pitch range, respectively. It was, thus, concluded that medium-sized leaf-cutting ant workers produced 10 significantly different acoustic signals depending on the situation and stimuli.
Accordingly, acoustic communication in fungus-growing ant societies was found to be more important than previously assumed. The evolutionary nature of ants was further revealed by two findings: the frequency of acoustic signal production increased with social evolutionary stage, and the complex society of leaf-cutting ants produced the highest number of eusocial insect sound types recorded thus far. Accordingly, acoustic forms of communication, much like chemical communication, are an important factor in the evolution and maintenance of social system in the ants, as ‘chattier’ communities appear capable of more complex societies than ‘silent’, small, and simple societies.
In leaf-cutting ants, there were 10 types of stridulatory sounds identified in the same caste, representing the highest number among eusocial insects such as bees (Apis mellifera25, 7; A. cerana26, 3; and other bees26, 1–3 types) and termites27 (higher termites: Constrictotermes cyphergaster28, 1 and Macrotermes natalensis29, 3; lower termites: Mastotermes darwiniensis30, 2). Termites, including fungus-growing agriculture-based species, are known to use drumming sounds as alarm signals23; however, there is no further evidence of complex acoustic signals for communication among insects.
The honeybee is a eusocial insect that is capable of complex acoustic communication despite the absence of a specific sound producing organ. They can produce vibroacoustic signals by shaking their thoraxes and wing vibrations31, and when workers combine figure-eight dances with acoustic signals, they can share information about both the distance and direction of nectar sources31. The tooting and quacking sounds of queen bees inhibit the hatching of new queens and inform nestmates of the presence and activity of enclosed queens32. In worker bees, a piping sound terminates the dance and helps recruit nectar receivers33. Alternatively, the leaf-cutting ants here demonstrated 10 significantly different acoustic signals, indicating a potentially more complex communication system.
A subset of ant species are the only eusocial insects with stridulatory organs to produce sound. Only groups with two petiole segments have these organs, comprising just six of the 20 subfamilies, including Myrmecinae, Paraponerinae, and Ponerinae34. Although there is some ant sound communication research in several ant species, no quantitative studies have been conducted on these functions. Accordingly, the high-resolution recording device we developed could be useful in facilitating discovery of more complex acoustic communication systems within eusocial insect societies in the future.
Evolutionary relations of sound organs
Head width and the stridulatory organ area were measured for 69 individuals of seven genera (eight species) of attines using SEM, and the following allometric equation was derived: log(organ area) = 14.41·log(head width) – 19.54 (R2 = 0.847; Fig. 2a, Extended Data 5). When tested for Spearman’s rank correlation coefficient, a significant positive relationship was observed between the stridulatory organ area and body size, with an S-value of 10,752 (P < 0.0001). The relationship between head width and slit number was also calculated: log(slit number) = 4.67·log(head width) – 5.37 (R2 = 0.61; S-value = 23,899; P < 0.0001; Fig. 2b). More specifically, the area of the stridulatory organ was not correlated with head width in the ancestral and middle-advanced groups but highly correlated in the two genera of leaf-cutting ants. The slopes (α) were 12.66 for Atta colombica (S-value = 80.71; P < 0.001) and 1.01 for Acromyrmex octospinosus (S-value = 514; P < 0.001), whereas the intercepts (β) were -15.73 and 1.94, respectively. Similarly, no significant correlation was found between the allometry of observed slit number and the ancestral or middle-advanced groups, although a significant positive correlation with Atta (α = 3.18, β = -2.19; S-value = 138.12; P < 0.01) and Acromyrmex was observed (α = 1.09, β = 1.51; S-value = 503.58; P < 0.001). The data thus suggest that even among two advanced genera, Acromyrmex followed a relative growth curve, whereas the more advanced genus Atta had slopes much greater than 1, indicating a highly variable structure.
These observations prompted an assumption regarding the physical evolution of acoustic communication in ants. The allometry indicated that the area and slit numbers of the stridulatory organ increased with body size. Body size, in turn, was regulated by habitat, queen egg-laying ability, and colony size6, suggesting that such ecological and social changes had driven the evolution of communication ability in ants.
In Hymenoptera, allometric analyses have shown that the slope of their reproductive organs is significantly >1, whereas those for the brain and central nervous system are <135. Reproductive organ size is correlated with egg size, and egg size depends on the presence of parasitic organisms; thus, changes in the allometric slope can indicate an adaptive pathway for external factors. This suggests that the stridulatory organs of Atta were subjected to positive selection pressure, significantly increasing their function as a critical means of communication. In turn, it may affect the ecological factors and account for the 100-fold greater colony size and more complex social structure of Atta than of Acromyrmex.
Chemical vs. acoustic cues
To compare the efficiency of social behaviours and management among leaf-cutting ants, sound and pheromone inhibition experiments were conducted. Ten sub-colonies, each consisting of eight workers, were placed for 1 week near a small fungus garden, along with glued pheromone-producing organs, sound-producing organs, or upper part of the mesothorax. Daily garden weights and social behaviours were recorded. The mortality rates in each experimental treatment were negligible and did not significantly affect the experimental results. It was found that the fungus garden was significantly smaller in the sound-inhibition group (48.4% ± 9.2% of the initial garden size) than in the pheromone-inhibition group (71.6% ± 10.9% of the initial garden size; Extended Data 6); the control garden was 103% ± 9.19% of the initial garden size. Significant differences among the groups were observed using a multiple comparison test (Steel–Dwass method: control vs. sound, t = 3.81; P < 0.001; sound vs. pheromone, t = 3.66; P < 0.001; control vs. pheromone, t = 3.70; P < 0.001).
Additionally, a behaviour comparison revealed that each inhibition group showed decreased fungus garden maintenance (FG), gathering of vegetal substrate (FOOD), dumping of garden waste pieces (DUMPING), nest digging (DIGGING), defensive behaviours (GUARD), and walking around (WALK) (Fig. 3). A generalized linear model analysis indicated that the behaviours with a significant effect on fungus garden maintenance were FG, FOOD, DUMP, and GUARD, (best model: FG, P < 0.01; FOOD, P < 0.01; DUMP, P < 0.0001; GUARD, P < 0.01, df, 6, AIC = -148.79).
These data indicate a reversal of conventional beliefs regarding ant communication, as chemical communication has historically been considered the most effective factor supporting intimate social behaviour, with all other factors being supplementary; the results here indicate that acoustic communication in leaf-cutting ants more effectively regulates social and cooperative behaviours.