Genomic Signatures of Domestication in a Fungus Obligately Farmed by Leafcutter Ants.

The naturally selected fungal crop (Leucoagaricus gongylophorus) farmed by leafcutter ants shows striking parallels with artificially selected plant crops domesticated by humans (e.g. polyploidy, engorged nutritional rewards, and dependence on cultivation). To date, poorly resolved L. gongylophorus genome assemblies based on short-read sequencing have constrained hypotheses about how millions of years under cultivation by ants shaped the fungal crop genome and potentially drove domestication. We use PacBio HiFi sequencing of L. gongylophorus from the leafcutter ant Atta colombica to identify 18 putatively novel biosynthetic gene clusters that likely cemented life as a cultivar (e.g. plant fragment degradation, ant-farmer communication, and antimicrobial defense). Comparative analyses with cultivated and free-living fungi showed genomic signatures of stepwise domestication transitions: (i) free-living to ant-cultivated: loss of genes conferring stress response and detoxification; (ii) hyphal food to engorged nutritional rewards: expansions of genes governing cellular homeostasis, carbohydrate metabolism, and siderophore biosynthesis; and (iii) detrital provisioning to freshly cut plant fragments: gene expansions promoting cell wall biosynthesis, fatty acid metabolism, and DNA repair. Comparisons across L. gongylophorus fungi farmed by 3 leafcutter ant species highlight genomic signatures of exclusively vertical clonal propagation and widespread transposable element activity. These results show how natural selection can shape domesticated cultivar genomes toward long-term ecological resilience of farming systems that have thrived across millennia.

The evolution of thermal performance curves in fungi farmed by attine ant mutualists in aboveground or belowground microclimates.

Fungi are abundant and ecologically important at a global scale, but little is known about whether their thermal adaptations are shaped by biochemical constraints (i.e. the Hotter is Better Model, HBM) or evolutionary tradeoffs (i.e., the Specialist Generalist Model, SGM). We tested these hypotheses by generating thermal performance curves (TPCs) of fungal cultivars farmed by six species of Panamanian fungus-farming 'attine' ants. These fungi represent evolutionary transitions in farming strategies as four cultivars are farmed by ants belowground at stable temperatures near 25°C and two cultivars are farmed aboveground at variable temperatures. We generated TPCs using a common garden experiment confining fungal isolates to different temperatures and then used a Bayesian hierarchical modeling approach to compare competing temperature sensitivity models. Some thermal performance traits differed consistently across farming strategies, with aboveground cultivars having: 1) higher tolerance to low temperatures (CTLmin) and 2) higher maximum growth rate at the optimal temperature (rmax). However, two core assumptions shared by the HBM or SGM were not supported as aboveground cultivars did not show systematic increases in either their optimal temperature (Topt) or thermal tolerance breadth. These results harness ant farming systems as long-term natural experiments to decouple the effects of environmental thermal variation and innate physiological temperature sensitivity on fungal thermal evolution. The results have clear implications for predicting climate warming induced breaking points in animal-microbe mutualisms.

A fungal symbiont converts provisioned cellulose into edible yield for its leafcutter ant farmers.

While ants are dominant consumers in terrestrial habitats, only the leafcutters practice herbivory. Leafcutters do this by provisioning a fungal cultivar (Leucoagaricus gongylophorus) with freshly cut plant fragments and harnessing its metabolic machinery to convert plant mulch into edible fungal tissue (hyphae and swollen hyphal cells called gongylidia). The cultivar is known to degrade cellulose, but whether it assimilates this ubiquitous but recalcitrant molecule into its nutritional reward structures is unknown. We use in vitro experiments with isotopically labelled cellulose to show that fungal cultures from an Atta colombica leafcutter colony convert cellulose-derived carbon into gongylidia, even when potential bacterial symbionts are excluded. A laboratory feeding experiment showed that cellulose assimilation also occurs in vivo in A. colombica colonies. Analyses of publicly available transcriptomic data further identified a complete, constitutively expressed, cellulose-degradation pathway in the fungal cultivar. Confirming leafcutters use cellulose as a food source sheds light on the eco-evolutionary success of these important herbivores.

The multidimensional nutritional niche of fungus-cultivar provisioning in free-ranging colonies of a neotropical leafcutter ant.

Foraging trails of leafcutter colonies are iconic scenes in the Neotropics, with ants collecting freshly cut plant fragments to provision a fungal food crop. We hypothesised that the fungus-cultivar's requirements for macronutrients and minerals govern the foraging niche breadth of Atta colombica leafcutter ants. Analyses of plant fragments carried by foragers showed how nutrients from fruits, flowers and leaves combine to maximise cultivar performance. While the most commonly foraged leaves delivered excess protein relative to the cultivar's needs, in vitro experiments showed that the minerals P, Al and Fe may expand the leafcutter foraging niche by enhancing the cultivar's tolerance to protein-biased substrates. A suite of other minerals reduces cultivar performance in ways that may render plant fragments with optimal macronutrient blends unsuitable for provisioning. Our approach highlights how the nutritional challenges of provisioning a mutualist can govern the multidimensional realised niche available to a generalist insect herbivore.

Nutrition mediates the expression of cultivar-farmer conflict in a fungus-growing ant.

Attine ants evolved farming 55-60 My before humans. Although evolutionarily derived leafcutter ants achieved industrial-scale farming, extant species from basal attine genera continue to farm loosely domesticated fungal cultivars capable of pursuing independent reproductive interests. We used feeding experiments with the basal attine Mycocepurus smithii to test whether reproductive allocation conflicts between farmers and cultivars constrain crop yield, possibly explaining why their mutualism has remained limited in scale and productivity. Stoichiometric and geometric framework approaches showed that carbohydrate-rich substrates maximize growth of both edible hyphae and inedible mushrooms, but that modest protein provisioning can suppress mushroom formation. Worker foraging was consistent with maximizing long-term cultivar performance: ant farmers could neither increase carbohydrate provisioning without cultivars allocating the excess toward mushroom production, nor increase protein provisioning without compromising somatic cultivar growth. Our results confirm that phylogenetically basal attine farming has been very successful over evolutionary time, but that unresolved host-symbiont conflict may have precluded these wild-type symbioses from rising to ecological dominance. That status was achieved by the evolutionarily derived leafcutter ants following full domestication of a coevolving cultivar 30-35 Mya after the first attine ants committed to farming.

Disentangling nutritional pathways linking leafcutter ants and their co-evolved fungal symbionts using stable isotopes.

Leafcutter ants are the ultimate insect superorganisms, with up to millions of physiologically specialized workers cooperating to cut and transport vegetation and then convert it into compost used to cultivate co-evolved fungi, domesticated over millions of years. We tested hypotheses about the nutrient-processing dynamics governing this functional integration, tracing 15 N- and 13 C-enriched substrates through colonies of the leafcutter ant Atta colombica. Our results highlight striking performance efficiencies, including rapid conversion (within 2 d) of harvested nutrients into edible fungal tissue (swollen hyphal tips called gongylidia) in the center of fungus gardens, while also highlighting that much of each colony's foraging effort resulted in substrate placed directly in the trash. We also find nutrient-specific processing dynamics both within and across layers of the fungus garden, and in ant consumers. Larvae exhibited higher overall levels of 15 N and 13 C enrichment than adult workers, supporting that the majority of fungal productivity is allocated to colony growth. Foragers assimilated 13 C-labeled glucose during its ingestion, but required several days to metabolically process ingested 15 N-labeled ammonium nitrate. This processing timeline helps resolve a 40-yr old hypothesis, that foragers (but apparently not gardeners or larvae) bypass their fungal crops to directly assimilate some of the nutrients they ingest outside the nest. Tracing these nutritional pathways with stable isotopes helps visualize how physiological integration within symbiotic networks gives rise to the ecologically dominant herbivory of leafcutter ants in habitats ranging from Argentina to the southern United States.

Metabolism and the rise of fungus cultivation by ants.

Most ant colonies are comprised of workers that cooperate to harvest resources and feed developing larvae. Around 50 million years ago (MYA), ants of the attine lineage adopted an alternative strategy, harvesting resources used as compost to produce fungal gardens. While fungus cultivation is considered a major breakthrough in ant evolution, the associated ecological consequences remain poorly understood. Here, we compare the energetics of attine colony-farms and ancestral hunter-gatherer colonies using metabolic scaling principles within a phylogenetic context. We find two major energetic transitions. First, the earliest lower-attine farmers transitioned to lower mass-specific metabolic rates while shifting significant fractions of biomass from ant tissue to fungus gardens. Second, a transition 20 MYA to specialized cultivars in the higher-attine clade was associated with increased colony metabolism (without changes in garden fungal content) and with metabolic scaling nearly identical to hypometry observed in hunter-gatherer ants, although only the hunter-gatherer slope was distinguishable from isometry. Based on these evolutionary transitions, we propose that shifting living-tissue storage from ants to fungal mutualists provided energetic storage advantages contributing to attine diversification and outline critical assumptions that, when tested, will help link metabolism, farming efficiency, and colony fitness.

Ecological strategies of (pl)ants: Towards a world-wide worker economic spectrum for ants.

Current global challenges call for a rigorously predictive ecology. Our understanding of ecological strategies, imputed through suites of measurable functional traits, comes from decades of work that largely focussed on plants. However, a key question is whether plant ecological strategies resemble those of other organisms.Among animals, ants have long been recognised to possess similarities with plants: as (largely) central place foragers. For example, individual ant workers play similar foraging roles to plant leaves and roots and are similarly expendable. Frameworks that aim to understand plant ecological strategies through key functional traits, such as the 'leaf economics spectrum', offer the potential for significant parallels with ant ecological strategies.Here, we explore these parallels across several proposed ecological strategy dimensions, including an 'economic spectrum', propagule size-number trade-offs, apparency-defence trade-offs, resource acquisition trade-offs and stress-tolerance trade-offs. We also highlight where ecological strategies may differ between plants and ants. Furthermore, we consider how these strategies play out among the different modules of eusocial organisms, where selective forces act on the worker and reproductive castes, as well as the colony.Finally, we suggest future directions for ecological strategy research, including highlighting the availability of data and traits that may be more difficult to measure, but should receive more attention in future to better understand the ecological strategies of ants. The unique biology of eusocial organisms provides an unrivalled opportunity to bridge the gap in our understanding of ecological strategies in plants and animals and we hope that this perspective will ignite further interest. Read the free Plain Language Summary for this article on the Journal blog.

Towards a general life-history model of the superorganism: predicting the survival, growth and reproduction of ant societies.

Social insect societies dominate many terrestrial ecosystems across the planet. Colony members cooperate to capture and use resources to maximize survival and reproduction. Yet, when compared with solitary organisms, we understand relatively little about the factors responsible for differences in the rates of survival, growth and reproduction among colonies. To explain these differences, we present a mathematical model that predicts these three rates for ant colonies based on the body sizes and metabolic rates of colony members. Specifically, the model predicts that smaller colonies tend to use more energy per gram of biomass, live faster and die younger. Model predictions are supported with data from whole colonies for a diversity of species, with much of the variation in colony-level life history explained based on physiological traits of individual ants. The theory and data presented here provide a first step towards a more general theory of colony life history that applies across species and environments.

A life history continuum in the males of a Neotropical ant assemblage: refuting the sperm vessel hypothesis.

Animal lifespans range from a few days to many decades, and this life history diversity is especially pronounced in ants. Queens can live for decades. Males, in contrast, are often assumed to act as ephemeral sperm delivery vessels that die after a brief mating flight-a view developed from studies of lekking species in temperate habitats. In a tropical ant assemblage, we found that males can live days to months outside the nest, a trait hypothesized to be associated with female calling, another common mating system. We combined feeding experiments with respirometry to show that lifespan can be enhanced over 3 months by feeding outside the nest. In one focal female calling species, Ectatomma ruidum, feeding enhanced male lifespan, but not sperm content. Extended lifespans outside the nest suggest stronger than expected selection on premating traits of male ants, although the ways these traits shape male mating success remain poorly understood.

Effect of scattered and discrete hydramethylnon bait placement on the Asian needle ant.

The Asian needle ant (Pachycondyla chinensis Emery) is invading natural and disturbed habitats across the eastern United States. While recent studies document the impact of P. chinensis on native ecosystems and human health, effective control measures remain unknown. Thus, we evaluated the field performance of a hydramethylnon granular bait, Maxforce Complete Granular Insect Bait, dispersed in clumps or scattered against P. chinensis. We also measured the effect of this bait on P. chinensis outside of the treatment zone. Surprisingly, unlike reports for other ant species, we achieved nearly complete P. chinensis population reductions 1 d after treatments were applied. Significant ant reductions were achieved until the end of our study at 28 d. No difference was recorded between clumped and scattered application methods. We found no overall difference in ant reductions from the edge out to 5 m beyond the treatment zone. Other local ant species appeared to be unaffected by the bait and foraging activity increased slightly after P. chinensis removal from treated areas. We suggest that Maxforce Complete Granular Insect Bait can be effective in an Asian needle ant treatment program.

Orthogonal protocols for DNA extraction from filamentous fungi.

There are few protocols available for DNA extraction from fungi. Here we present four complementary protocols for extraction of genomic DNA from fungi. We quantify the efficacy of extractions and compare eight species from five filamentous fungal genera, including both basidiomycetes and ascomycetes. These protocols should be useful for extraction of DNA from a variety of filamentous fungi. For complete details on the use and execution of this protocol, please refer to Conlon et al. (2021).

Nutritional challenges of feeding a mutualist: Testing for a nutrient-toxin tradeoff in fungus-farming leafcutter ants.

The biochemical heterogeneity of food items often yields tradeoffs as each bite of food tends to contain some nutrients in surplus and others in deficit, as well as other less palatable or even toxic compounds. These multidimensional nutritional challenges are likely to be compounded when foraged foods are used to provision others (e.g., offspring or symbionts) with different physiological needs and tolerances. We explored these challenges in free-ranging colonies of leafcutter ants that navigate a diverse tropical forest to collect plant fragments they use to provision a co-evolved fungal cultivar. We tested the prediction that leafcutter farmers face provisioning tradeoffs between the nutritional quality and concentration of toxic tannins in foraged plant fragments. Chemical analyses of plant fragments sampled from the mandibles of Panamanian Atta colombica leafcutter ants provided little support for a nutrient-tannin foraging tradeoff. First, colonies foraged for plant fragments that ranged widely in tannin concentration. Second, high tannin levels did not appear to restrict colonies from selecting plant fragments with blends of protein and carbohydrates that maximized cultivar performance when measured with in vitro experiments. We also tested whether tannins expand the realized nutritional niche selected by leafcutter ants into high-protein dimensions as: (1) tannins can bind proteins and reduce their accessibility during digestion, and (2) in vitro experiments have shown that excess protein provisioning reduces cultivar performance. Contrary to this hypothesis, the most protein-rich plant fragments did not have highest tannin levels. More generally, the approach developed here can be used to test how multidimensional interactions between nutrients and toxins shape the costs and benefits of providing care to offspring or symbionts.

Male ant reproductive investment in a seasonal wet tropical forest: Consequences of future climate change.

Tropical forests sustain many ant species whose mating events often involve conspicuous flying swarms of winged gynes and males. The success of these reproductive flights depends on environmental variables and determines the maintenance of local ant diversity. However, we lack a strong understanding of the role of environmental variables in shaping the phenology of these flights. Using a combination of community-level analyses and a time-series model on male abundance, we studied male ant phenology in a seasonally wet lowland rainforest in the Panama Canal. The male flights of 161 ant species, sampled with 10 Malaise traps during 58 consecutive weeks (from August 2014 to September 2015), varied widely in number (mean = 9.8 weeks, median = 4, range = 1 to 58). Those species abundant enough for analysis (n = 97) flew mainly towards the end of the dry season and at the start of the rainy season. While litterfall, rain, temperature, and air humidity explained community composition, the time-series model estimators elucidated more complex patterns of reproductive investment across the entire year. For example, male abundance increased in weeks when maximum daily temperature increased and in wet weeks during the dry season. On the contrary, male abundance decreased in periods when rain receded (e.g., at the start of the dry season), in periods when rain fell daily (e.g., right after the beginning of the wet season), or when there was an increase in the short-term rate of litterfall (e.g., at the end of the dry season). Together, these results suggest that the BCI ant community is adapted to the dry/wet transition as the best timing of reproductive investment. We hypothesize that current climate change scenarios for tropical regions with higher average temperature, but lower rainfall, may generate phenological mismatches between reproductive flights and the adequate conditions needed for a successful start of the colony.

Nutritional niches reveal fundamental domestication trade-offs in fungus-farming ants.

During crop domestication, human farmers traded greater productivity for higher crop vulnerability outside specialized cultivation conditions. We found a similar domestication trade-off across the major co-evolutionary transitions in the farming systems of attine ants. First, the fundamental nutritional niches of cultivars narrowed over ~60 million years of naturally selected domestication, and laboratory experiments showed that ant farmers representing subsequent domestication stages strictly regulate protein harvest relative to cultivar fundamental nutritional niches. Second, ants with different farming systems differed in their abilities to harvest the resources that best matched the nutritional needs of their fungal cultivars. This was assessed by quantifying realized nutritional niches from analyses of items collected from the mandibles of laden ant foragers in the field. Third, extensive field collections suggest that among-colony genetic diversity of cultivars in small-scale farms may offer population-wide resilience benefits that species with large-scale farming colonies achieve by more elaborate and demanding practices to cultivate less diverse crops. Our results underscore that naturally selected farming systems have the potential to shed light on nutritional trade-offs that shaped the course of culturally evolved human farming.

Nutritional Dimensions of Invasive Success.

Despite mounting calls for predictive ecological approaches rooted in physiological performance currencies, the field of invasive species biology has lagged behind. For instance, successful invaders are often predicted to consume diverse foods, but the nutritional complexity of foods often leaves food-level analyses short of physiological mechanisms. The emerging field of nutritional geometry (NG) provides new theory and empirical tools to predict invasive potential based on fundamental and realized nutritional niches. We review recent advances and synthesize NG predictions about behavioral traits that favor invasive establishment, and evolutionary dynamics that promote invasive spread. We also provide practical advice for applying NG approaches, and discuss the power of nutrition to achieve a more predictive invasion biology that explicitly integrates physiological mechanisms.

Using Nutritional Geometry to Explore How Social Insects Navigate Nutritional Landscapes.

Insects face many cognitive challenges as they navigate nutritional landscapes that comprise their foraging environments with potential food items. The emerging field of nutritional geometry (NG) can help visualize these challenges, as well as the foraging solutions exhibited by insects. Social insect species must also make these decisions while integrating social information (e.g., provisioning kin) and/or offsetting nutrients provisioned to, or received from unrelated mutualists. In this review, we extend the logic of NG to make predictions about how cognitive challenges ramify across these social dimensions. Focusing on ants, we outline NG predictions in terms of fundamental and realized nutritional niches, considering when ants interact with related nestmates and unrelated bacterial, fungal, plant, and insect mutualists. The nutritional landscape framework we propose provides new avenues for hypothesis testing and for integrating cognition research with broader eco-evolutionary principles.

Using nutritional geometry to define the fundamental macronutrient niche of the widespread invasive ant Monomorium pharaonis.

The emerging field of nutritional geometry (NG) provides powerful new approaches to test whether and how organisms prioritize specific nutritional blends when consuming chemically complex foods. NG approaches can thus help move beyond food-level estimates of diet breadth to predict invasive success, for instance by revealing narrow nutritional niches if broad diets are actually composed of nutritionally similar foods. We used two NG paradigms to provide different, but complementary insights into nutrient regulation strategies and test a hypothesis of extreme nutritional generalism in colony propagules of the globally distributed invasive ant Monomorium pharaonis. First, in two dimensions (protein:carbohydrates; P:C), M. pharaonis colonies consistently defended a slightly carbohydrate-biased intake target, while using a generalist equal-distance strategy of collectively overharvesting both protein and carbohydrates to reach this target when confined to imbalanced P:C diets. Second, a recently developed right-angled mixture triangle method enabled us to define the fundamental niche breadth in three dimensions (protein:carbohydrates:lipid, P:C:L). We found that colonies navigated the P:C:L landscape, in part, to mediate a tradeoff between worker survival (maximized on high-carbohydrate diets) and brood production (maximized on high-protein diets). Colonies further appeared unable to avoid this tradeoff by consuming extra lipids when the other nutrients were limiting. Colonies also did not rely on nutrient regulation inside their nests, as they did not hoard or scatter fractions of harvested diets to adjust the nutritional blends they consumed. These complementary NG approaches highlight that even the most successful invasive species with broad fundamental macronutrient niches must navigate complex multidimensional nutritional landscapes to acquire limiting macronutrients and overcome developmental constraints as small propagules.

Reconstructing the functions of endosymbiotic Mollicutes in fungus-growing ants.

Mollicutes, a widespread class of bacteria associated with animals and plants, were recently identified as abundant abdominal endosymbionts in healthy workers of attine fungus-farming leaf-cutting ants. We obtained draft genomes of the two most common strains harbored by Panamanian fungus-growing ants. Reconstructions of their functional significance showed that they are independently acquired symbionts, most likely to decompose excess arginine consistent with the farmed fungal cultivars providing this nitrogen-rich amino-acid in variable quantities. Across the attine lineages, the relative abundances of the two Mollicutes strains are associated with the substrate types that foraging workers offer to fungus gardens. One of the symbionts is specific to the leaf-cutting ants and has special genomic machinery to catabolize citrate/glucose into acetate, which appears to deliver direct metabolic energy to the ant workers. Unlike other Mollicutes associated with insect hosts, both attine ant strains have complete phage-defense systems, underlining that they are actively maintained as mutualistic symbionts.

Cryptic Diversity in Colombian Edible Leaf-Cutting Ants (Hymenoptera: Formicidae).

Leaf-cutting ants are often considered agricultural pests, but they can also benefit local people and serve important roles in ecosystems. Throughout their distribution, winged reproductive queens of leaf-cutting ants in the genus Atta Fabricius, 1804 are consumed as a protein-rich food source and sometimes used for medical purposes. Little is known, however, about the species identity of collected ants and the accuracy of identification when ants are sold, ambiguities that may impact the conservation status of Atta species as well as the nutritional value that they provide to consumers. Here, 21 samples of fried ants bought in San Gil, Colombia, were identified to species level using Cytochrome Oxidase I (COI) barcoding sequences. DNA was extracted from these fried samples using standard Chelex extraction methods, followed by phylogenetic analyses with an additional 52 new sequences from wild ant colonies collected in Panama and 251 publicly available sequences. Most analysed samples corresponded to Atta laevigata (Smith, 1858), even though one sample was identified as Atta colombica Guérin-Méneville, 1844 and another one formed a distinct branch on its own, more closely related to Atta texana (Buckley, 1860) and Atta mexicana (Smith, 1858). Analyses further confirm paraphyly within Atta sexdens (Linnaeus, 1758) and A. laevigata clades. Further research is needed to assess the nutritional value of the different species.