## 6 Impacts of Leaf-Cutter Ants on Biogeochemical Cycles  
Leaf-cutter ants are geographically widespread and abundant in lowland neotropical forests where they are \"ecosystem engineers\" with a significant effect on the ecosystem processes of carbon and nitrogen cycling (Figure 7). They account for as much as one quarter of all herbivores in these forests, and their symbionts may be responsible for significant heterogeneity in soil nitrogen resources (Figure 5). Through their herbivory, leaf-cutter ants change their environment by creating canopy gaps, transferring organic matter underground, enhancing soil aeration and turnover rates, and increasing soil nutrient availability and nitrogen fixation [35; 36; 37; 38]. Leaf-cutter ants transport substantial amounts of organic material into their nests and increase the soil organic matter pool that fungi and bacteria transform into gaseous and aqueous phases. Their nests appear as mounds of excavated soil marked by numerous entrances and gas vents that lead to an intricate network of tunnels and chambers. Using the plant material they collect, leaf-cutter ants cultivate an obligate symbiotic fungus, which  
Figure 7: Image from the automated minithizotron (AMR) system showing a suberizing fine root, soil aggregates, and arbuscular mycorrhizal fungal hyphal networks. Photo by [PERSON]. they consume as food. Modifying the biotic and abiotic components in their habitats and regulating the availability of resources, they influence other species. The combined concentration of plant material with fungal and ant activity is high. This activity makes leaf-cutter nests hot spots for biogeochemical cycling [39; 40]. Their nests alter the physical and chemical properties of soils, and the fungus gardens inside the nests are sites of symbiotic N\({}_{2}\) fixation [38]. The roots and fungal hyphae preferentially grow inside the nests and their turnover contributes to the soil carbon pool. All these processes directly fertilize the nest soils and hypothetically promote CO\({}_{2}\) efflux. As forests become increasingly fragmented across the Neotropics because of land use changes due to agriculture and grazing, leaf-cutter ants are becoming more abundant [37; 40; 41] and their impact on soil carbon dynamics is expected to increase. Our research investigated the carbon dynamics as influenced by leaf-cutter ants and their nests and differentiated the sources of CO\({}_{2}\) efflux between the activities of ants, fungi, the nest microbial community, and roots and hyphae (Figure 8). Additionally, we quantified the nitrogen and phosphorus biogeochemistry of ant nests to determine their legacy effects after the nests are abandoned. This research involved a multifaceted approach using new technologies to couple continuous measurements of the soil CO\({}_{2}\) with discrete measures of the CH\({}_{4}\) efflux, as well as the isotopic \(\delta^{13}\)C composition, soil carbon pools and fluxes, soil nutrient concentrations, estimates of root and fungal dynamics, and microbial community and functional indices. The measurements were carried out on ant nests, paired control sites, and along a chronosequence of abandoned nests to quantify the legacy effects. The CO\({}_{2}\) concentrations in nest tunnels were higher than the background (atmospheric) levels and at times exceeded 5% (by volume) in the vents connected to the fungal and refuse chambers [42; 43; 44]. Thus, the nest soils adjacent to these tunnels were nutrient hot spots, emitting more CO\({}_{2}\) than the soils away from the nests. By continuously excavating the ventilation network in their nests, leaf-cutter ant colonies maintain adequate CO\({}_{2}\) and O\({}_{2}\) concentrations (Figure 8). Prior to our research, the CO\({}_{2}\) emission rates had not been well characterized, and the potential connection between the nest air and the surrounding nest soils had not been recognized (Figure 8).