Significance Flowering plants are thought to have facilitated ant diversification by providing novel sources of food and habitat, and ants to have facilitated plant diversification by providing defense and dispersal. We test these hypotheses by inferring when and how interactions between ants and plants evolved, and examine their macroevolutionary consequences in ants. Although plants with ant-associated traits have been shown to have higher diversification rates, here we demonstrate that living and feeding on plants does not affect ant diversification. Ants used plants as food and nesting sources substantially before the evolution of ant-associated structures. Arboreal foraging preceded use of plant-derived food sources. Arboreal nesting then largely evolved from arboreally foraging ancestors. Finally, omnivory served as an evolutionary link between predation and herbivory.

Abstract Ant–plant interactions are diverse and abundant and include classic models in the study of mutualism and other biotic interactions. By estimating a time-scaled phylogeny of more than 1,700 ant species and a time-scaled phylogeny of more than 10,000 plant genera, we infer when and how interactions between ants and plants evolved and assess their macroevolutionary consequences. We estimate that ant–plant interactions originated in the Mesozoic, when predatory, ground-inhabiting ants first began foraging arboreally. This served as an evolutionary precursor to the use of plant-derived food sources, a dietary transition that likely preceded the evolution of extrafloral nectaries and elaiosomes. Transitions to a strict, plant-derived diet occurred in the Cenozoic, and optimal models of shifts between strict predation and herbivory include omnivory as an intermediate step. Arboreal nesting largely evolved from arboreally foraging lineages relying on a partially or entirely plant-based diet, and was initiated in the Mesozoic, preceding the evolution of domatia. Previous work has suggested enhanced diversification in plants with specialized ant-associated traits, but it appears that for ants, living and feeding on plants does not affect ant diversification. Together, the evidence suggests that ants and plants increasingly relied on one another and incrementally evolved more intricate associations with different macroevolutionary consequences as angiosperms increased their ecological dominance.

Biotic interactions are ubiquitous, yet their evolutionary origins and macroevolutionary consequences are often poorly understood. Placing the evolutionary history of interacting lineages and their associated traits in a temporal framework can help reveal the reciprocal dynamics of coevolution by identifying the sequence and tempo of events in the evolution of interactions, and the influence of these events on lineage diversification. The diversification of numerous terrestrial lineages is attributed, at least in part, to the decline of the Mesozoic, gymnosperm-dominated vegetation and the rise of the Cenozoic, angiosperm-dominated flora (refs. 1 and 2 and therein). Rapid diversification is commonly associated with the exploitation of novel niches and resources (3, 4). Unique aspects of angiosperm reproductive and vegetative biology, coupled with increased ecosystem complexity, provided numerous novel niches and resources that interacting lineages could exploit, whereas the evolution of angiosperms with high transpiration capacities altered the climate and enabled the development and expansion of tropical rainforests (2).

Ants (Hymenoptera: Formicidae) are among the most abundant insects on Earth (5). Diversification of the major ant lineages increased during the global transition to angiosperm dominance (1, 6, 7), when ancestrally carnivorous ant lineages that occupied the soil or litter layer are thought to have incorporated plant-based food sources (PBFS) into their diet (8), setting the stage for their expansion into arboreal habitats and previously unexploited ecosystems (9). The increased ecological opportunity provided may therefore have enhanced diversification of lineages able to exploit these resources. In addition, acquisition of plant-derived food sources may be more metabolically efficient, as these sedentary food sources do not require energy to be expended on subduing prey (10). As elevated diversification rates have been inferred in various herbivorous and arboreal clades of animals and epiphytic plants (11⇓⇓⇓⇓⇓–17), we thus predict that ant lineages that rely on plants for food and habitat may also show evidence of faster diversification (cf. ref. 18).

Ants interact with a range of tissues and structures across vascular plants, with the strength of these interactions varying from diffuse to highly specific (19⇓⇓⇓–23). The three most common structures are elaiosomes, extrafloral nectaries (EFNs), and domatia. Elaiosomes are lipid-rich seed appendages found in more than 11,000 species across 77 families of angiosperms, which ants use as a food source while protecting and dispersing the seeds to nutrient-rich locations and reducing parent–offspring competition (22⇓–24). EFNs (including foliar nectaries in ferns) occur in nearly 4,000 species in more than 100 families of angiosperms and ferns, providing nutrition to ants, whereas the ants protect the host plant from herbivores and pathogens (19, 21). Domatia are known from nearly 700 vascular plant species in 50 families and are specialized structures in which ants live and defend the host plant (20, 25). Ants may also obtain food from plants indirectly, by farming hemipterans for their liquid exudates (honeydew), often within these domatia (26⇓–28). The ecological advantages provided to plants through their interactions with ants may therefore be expected to confer success over macroevolutionary timescales. Previous work has yielded substantial insight, suggesting that EFN and elaiosome-forming plant lineages are associated with enhanced diversification rates (22, 29), whereas domatia formation is not expected to influence diversification rate (20). The fossil record of ants and plants is abundant and diverse, but their interactions are rarely preserved; as a consequence, comparative analyses of time-calibrated phylogenies may clarify their origins and interactions. Fossils reveal that EFNs and ant–hemipteran associations occurred as early as the Eocene (30, 31). Molecular clock analyses suggest that plant clades forming EFNs evolved by the Eocene (32⇓–34), Late Cretaceous origins of elaiosome-bearing families (23, 35), and Neogene origins of domatia (20). However, the coarse phylogenetic granularity (family-level) or the restriction to specific traits or clades leaves open many questions about the age and evolutionary pathways through which ant–plant interactions evolved.

Here we investigate the evolution of these interactions at a broad phylogenetic scale with a variety of analyses that complement earlier work. Specifically, we ask whether increased diversification in ants is associated with use of plants as food sources or habitat. We also investigate when plants evolved the potential to form ant-associated structures, both in geological time and in relation to the origin of plant use by ants.

Discussion Evolutionary Use of PBFS by Ants. We infer that arboreal foraging in ants preceded the transition from a strict predatory habit to a diet including plant-derived food sources, implying that these food sources were collected from the plant itself (including arboreal tending of sap-sucking insects), rather than from fallen plant tissue. Ant use of plants for food likely preceded the evolution of specialized food structures (EFNs and elaiosomes) in plants. These “early adopters” of the arboreal habitat may have first begun feeding on sap from cuts and wounds in the vegetation (41). In addition, many ants use hemipterans both for their honeydew as well as for prey (42); trophobiotic interactions between ants and aphids are thought to have originated as predator–prey relationships, which transitioned to mutualisms when aphids offered honeydew as a food source (43), and the same may apply to other ant–hemipteran interactions. Sap-sucking hemipterans evolved during or before the end of the Triassic (ref. 44 and therein), substantially predating ants, and the phylogenetic position of many trophobiotic clades (42, 45) is loosely consistent with early omnivore ant lineages relying partially on honeydew. It thus seems reasonable to suspect that early ants first transitioned to omnivory by opportunistically using sap from plant wounds and/or hemipteran honeydew before shifting fully to herbivory, as plants evolved specialized food sources for ants in return for defense and dispersal. Evolution of Obligate Herbivory. It remains unclear why it took more than 50 Ma for ants to specialize entirely on plant-derived food sources. Alternate dietary codings consistently revealed a relatively late transition to a strictly herbivorous diet (additional files in the Dryad repository). Our analyses suggest that several ant clades evolved a strict reliance on plant-associated food sources from omnivorous ancestors. The first to do so was a myrmicine subclade of fungus-farming ants (SI Appendix, Figs. S4–S8), whose adults feed on leaf sap and adults and juveniles on fungi (ref. 46 and therein); the transition was thus not facilitated by EFNs or elaiosomes. Several physiological challenges are encountered when foraging in exposed locations and consuming a plant-based diet, which may have imposed significant evolutionary constraints. In addition, food sources such as sap from plant wounds and hemipteran honeydew may have not been limiting until consumers achieved high abundance; the subsequent evolution of specialized food sources, such as EFNs and elaiosomes, may have then enabled certain omnivorous lineages to evolve a strict reliance on plant-derived food sources. Evolution of Arboreal Nesting. Our reconstructions show an evolutionary sequence in which a strictly predatory, ground-inhabiting ant lineage first transitioned to arboreal foraging, then incorporated plant-based foods into their diet, and finally began nesting arboreally. In plants, we infer that domatia evolved rather long after the origins of arboreal nesting, near the start of the Cenozoic (Fig. 2; see also ref. 20). Several large, monophyletic clades of strict, arboreal nesting ants predate the evolution of domatia; thus, we anticipate this finding will remain robust to a refined, species-level coding. The reasons for this temporal mismatch are unclear. Although some have suggested that ants have nested in domatia since the Cretaceous (47), no fossil data confirm this supposition. Numerous extant, arboreally nesting ant species use unspecialized, preformed openings in trees or beneath epiphytic plants and their substrate, or construct their own nesting structures, such as carton or silk nests (48, 49). Thus, numerous options likely existed for arboreally nesting ants before the evolution of domatia, with recent work demonstrating that domatia nesting in acacia ants evolved from ancestors nesting in dead twigs (50). Domatia are thought to have evolved through a number of distinct pathways involving a range of plant organs, morphological traits, and even the presence of hemipterans, which are thought to have served as domatial precursors or predisposed plant lineages to their formation (20, 51, 52). The Age of Ant-Associated Traits in Plants. The phylogenetic resolution (genera) of our dataset limits the precision and certainty with which we can infer the origins of plant traits. Therefore, our binary scoring of the trait (EFNs, elaiosomes, or domatia) being present or not in at least one species of a genus means that our ASRs are best interpreted as an analysis of the potential for the trait to exist. Thus, the potential to form EFNs may have evolved as early as the mid-Cretaceous; however, it seems likely that their actual origin was much later. Chiefly, a rich record of vegetative plant fossils provides no evidence for the occurrence of EFNs before the late Eocene (30). Previous ancestral state estimates at the species level have shown that domatia and EFNs frequently evolved much later, typically within individual plant genera, and rarely are ancestral across larger, older clades (20, 29, 32). These analyses also showed that the presence of EFNs within a densely sampled clade is often marked by homoplasy. If the rate of gain and/or loss of EFNs has been consistently high in clades capable of producing them, they might have occurred ancestrally, but ephemerally. Our reconstruction of the evolutionary potential for these traits, although naive, nevertheless raises the possibility that EFNs and elaiosomes may have deeper ancestry than is commonly thought and motivates closer scrutiny of their evolution. Finally, the nonrandom distribution of these traits (here and refs. 21 and 29) suggests the presence of a genomic predisposition in these lineages enabling trait evolution, the presence of common selective agent precipitating their evolution, or a combination thereof. Diversification of Ants Using PBFS and Arboreal Habitats. Contrary to observations in many other organismal groups, use of plants for food or habitat does not confer enhanced diversification rates in ants. A number of factors may be responsible for these discrepancies. First, PBFS typically used by ants (nectar, honeydew) are low in nutritional value; as a consequence, these lineages may be competitively inferior or devote fewer resources to reproduction or dispersal. Occupation of arboreal habitats exposes ants to both increased physiological challenges (desiccation) and predation; as a consequence, these may have neutral or negative effects on fitness and not enhance diversification. More generally, diversification rates may have been elevated during initial exploitation of novel plant-based resources, but as resources were diminished and niches filled, increased competition may have hampered diversification, whereas priority effects linked to plant-based resources may produce nonuniform clade-level responses to independent invasions of a niche (53). Why Do Diversification Rate Patterns Differ Between Ants and Plants? Although theory and evidence suggest that biotic interactions are capable of influencing the dynamics of diversification, our results indicate that hypothesized beneficial effects on the fitness of interacting organisms may not be conferred to all interacting lineages at macroevolutionary scales (53). Benefits provided to plants by ants at the organismal level appear to translate to a macroevolutionary currency in which these plant lineages exhibit enhanced diversification rates (23, 29, 32). It is also worth considering the differences in how traits, our chosen indicators for ant–plant interactions, were scored for each group. For ants, plant-based food represents an amalgamation of food items that includes EFNs and elaiosomes, but also food bodies and hemipteran honeydew, among others. As a consequence, comparisons were not equivalent; for example, we were unable to strictly juxtapose diversification rates of elaiosome-feeding ants with elaiosome-producing plants. The ant coding scheme also included all ant taxa using plants; thus, mutualists and nonmutualists were included. In addition, phylogenetic scale can be important in detecting these processes. Plants may have facilitated diversification in specific lineages or windows of time, but they do not universally explain ant diversification, highlighting the ecological and evolutionary complexity of ant diversification. The Evolution of Ant–Plant Interactions. Here we suggest that although ant–plant interactions are characterized by temporal discordance, the evolution of these associations is likely tightly linked and evolved sequentially, but with asymmetric macroevolutionary consequences. Ants nested in plants and fed on plant-derived resources before the evolution of specialized plant structures such as EFNs, elaiosomes, and domatia, as would be expected. Nesting in plants before the evolution of domatia likely occurred in preexisting holes and gaps in plants or under epiphytes. The earliest omnivorous ants likely foraged arboreally and used plant sap and/or hemipteran honeydew as food sources. Ants relying strictly on a plant-based diet were derived from omnivorous lineages, with the earliest transition occurring more than 50 Ma after the origin of ants. Finally, although previous work suggests that plant lineages producing some of these ant-associated structures exhibited elevated diversification rates (23, 29, 32), our work demonstrates that ant lineages relying on plants for food, foraging, and nesting are not associated with enhanced diversification rates. This further suggests that biotic factors are capable of acting as agents of macroevolutionary change, but that the macroevolutionary consequences of these interactions cannot be universally predicted by effects at the organismic level (53). Furthermore, although ant–plant interactions originated in the Mesozoic, the evolution of specialized ant-associated structures and a strictly plant-derived diet likely evolved during the Cenozoic. Thus, as angiosperms achieved ecological dominance, ants and plants evolved more intricate associations with an increased interdependence on one another.

Methods Phylogeny: Formicidae. NCBI was queried for Formicidae accessions derived from 12 loci, and a stepwise alignment procedure guided by higher-level taxonomy was used to construct a global alignment. Alignments from individual loci were concatenated into a 9,714-bp alignment, and PartitionFinder (54) was employed to identify the optimal partitioning strategy as determined by the Bayesian Information Criterion (BIC). A partitioned ML analysis using the general time-reversible categorical (GTRCAT) approximation was performed using randomized axelerated maximum likelihood (RAxML) on the Cyberinfrastructure for Phylogenetic Research (CIPRES) cluster (55). Minimum age constraints were derived from a previous synthesis (56). Fifty-one fossils were included as minimum age constraints (SI Appendix, Table S7), and the root node, representing the crown node of Aculeata, was fixed to 185 Ma ago, as implemented in previous ant phylogenetic studies (57). The penalized likelihood algorithm, as implemented in treePL (58), was used to timescale the tree. ASR and Diversification Rate Analyses: Formicidae. Species richness estimates for extant genera were retrieved from AntWiki (www.antwiki.org/wiki/Species_Accounts), and subsequently used with our time-scaled phylogeny to estimate diversification rates across Formicidae in Bayesian analysis of macroevolutionary mixtures (59). Genus-specific sampling fractions were incorporated to account for incomplete sampling. Genus-level diet, foraging, and nesting locations were derived from a previous compilation (18), and character states applied to all species in a genus or genus-level group. Trait states in the original database were assigned to each genus based on literature records, and genera considered polymorphic if at least 10% of the examined species were in a conflicting state (18). Of the 1,731 ant taxa included, relatively few contained no information (diet, 67; foraging, 18; nesting, 20), whereas a larger number were polymorphic or ambiguous (diet, 245; foraging, 694; nesting, 589). Several coding schemes were created to accommodate taxa with questionable, missing, or multiple character states. In addition, dietary coding schemes with three states were also analyzed with a constrained transition matrix, which prevented direct transitions between herbivory and predation, forcing transitions to pass first through omnivory (“no02direct”). Marginal ancestral state estimates were then reconstructed using the “all rates different” (ARD) model in package corHMM (60) with the rayDISC function. We then also tested for the correlated evolution among diet, foraging, and nesting datasets, using the correlated path model (61) (a generalization of ref. 62) in corHMM (60) under the ARD model of evolution. We inferred when acquisitions of plant-associated traits occurred by dividing the tree into 1-Ma increments, and summing nonterminal nodes in which the most-likely state possessed the trait of interest and was descended from a node whose most-likely state lacked the trait of interest. As our interest was in documenting gains, rather than losses, this represents a cumulative total of gains of the trait through time. Summaries were made from the most-likely states derived from the best-fitting correlated paths model. We then tested for associations between character states and diversification rates in a number of ways, including a genus-level phylogenetic ANOVA (38), hidden state speciation and extinction analyses (63), and structured rate permutations on phylogenies analyses (39). Phylogeny: Plants. Streptophyte genus-level alignments (36) were used to infer a phylogeny of vascular plants. Higher-level taxonomy was used as a topological constraint, and a partitioned ML analysis run in ExaML (64), constraining the monophyly of clades at or above the rank of family. The resulting tree consisted of 10,785 genera, which was then made ultrametric in treePL, using the mean ages of shared nodes in the phylogeny of (65) as constraints. ASR Analyses: Plants. To identify plant genera that ants use as food sources (EFNs, elaiosomes) or form domatia, we used preexisting species or genus lists (20, 23, 66). These databases documented the presence of traits; as a consequence, taxa not included were scored as lacking the trait. Our phylogeny was linked to 84% (711/846), 86% (286/334), 90% (143/159) of genera known to possess EFNs, elaiosomes, and domatia, respectively. Any genus containing a species possessing these was coded as forming these (29), whereas genera not linked to trait databases were coded as lacking the trait. We measured phylogenetic signal [d-statistic (37)] in caper (67) and assessed significance through 1,000 permutations. ASR analyses were then conducted as earlier, using the ARD model. Associated files are included in the Dryad repository and GitHub, and additional details provided in the SI Appendix.

Acknowledgments We thank Benjamin Blanchard and Shauna Price for many helpful conversations and for reading a previous version to greatly improve this study. We also express our gratitude to the reviewers and handling editor for their constructive feedback, which greatly improved the manuscript. M.P.N. thanks the Negaunee Foundation Interdisciplinary Postdoctoral Fellowship at the Field Museum of Natural History for support. R.H.R. and C.S.M. thank the National Science Foundation for research support Awards (NSF EF-1208727 to R.H.R.; NSF DEB 1442316 and NSF IOS 1354193 to C.S.M.).

Footnotes Author contributions: M.P.N., R.H.R., and C.S.M. designed research; M.P.N. performed research; M.P.N. analyzed data; and M.P.N., R.H.R., and C.S.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

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