Co‐evolutionary patterns and diversification of ant–fungus associations in the asexual fungus‐farming ant Mycocepurus smithii in Panama

Partner fidelity through vertical symbiont transmission is thought to be the primary mechanism stabilizing cooperation in the mutualism between fungus‐farming (attine) ants and their cultivated fungal symbionts. An alternate or additional mechanism could be adaptive partner or symbiont choice mediating horizontal cultivar transmission or de novo domestication of free‐living fungi. Using microsatellite genotyping for the attine ant Mycocepurus smithii and ITS rDNA sequencing for fungal cultivars, we provide the first detailed population genetic analysis of local ant–fungus associations to test for the relative importance of vertical vs. horizontal transmission in a single attine species. M. smithii is the only known asexual attine ant, and it is furthermore exceptional because it cultivates a far greater cultivar diversity than any other attine ant. Cultivar switching could permit the ants to re‐acquire cultivars after garden loss, to purge inferior cultivars that are locally mal‐adapted or that accumulated deleterious mutations under long‐term asexuality. Compared to other attine ants, symbiont choice and local adaptation of ant–fungus combinations may play a more important role than partner‐fidelity feedback in the co‐evolutionary process of M. smithii and its fungal symbionts.     

Population genetic signatures of diffuse co-evolution between leaf-cutting ants and their cultivar fungi

Switching of symbiotic partners pervades most mutualisms, despite mechanisms that appear to enforce partner fidelity. To investigate the interplay of forces binding and dissolving mutualistic pairings, we investigated partner fidelity at the population level in the attine ant-fungal cultivar mutualism. The ants and their cultivars exhibit both broad-scale co-evolution, as well as cultivar switching, with short-term symbiont fidelity maintained by vertical transmission of maternal garden inoculates via dispersing queens and by the elimination of alien cultivar strains. Using microsatellite markers, we genotyped cultivar fungi associated with five co-occurring Panamanian attine ant species, representing the two most derived genera, leaf-cutters Atta and Acromyrmex. Despite the presence of mechanisms apparently ensuring the cotransmission of symbiont genotypes, different species and genera of ants sometimes shared identical fungus garden genotypes, indicating widespread cultivar exchange. The cultivar population was largely unstructured with respect to host ant species, with only 10% of the structure in genetic variance being attributable to partitioning among ant species and genera. Furthermore, despite significant genetic and ecological dissimilarity between Atta and Acromyrmex, generic difference accounted for little, if any, variance in cultivar population structure, suggesting that cultivar exchange dwarfs selective forces that may act to create co-adaptive ant-cultivar combinations. Thus, binding forces that appear to enforce host fidelity are relatively weak and pairwise associations between cultivar lineages and ant species have little opportunity for evolutionary persistence. This implicates that mechanisms other than partner fidelity feedback play important roles in stabilizing the leafcutter ant-fungus mutualism over evolutionary time.     

Ant versus fungus versus mutualism: ant-cultivar conflict and the deconstruction of the attine ant-fungus symbiosis

A century of research on fungus-growing ants (Attini, Formicidae) has ignored the cultivated fungi as passive domesticates and viewed the attine fungicultural symbiosis as an integrated unit dominated by the evolutionary interests of the ant farmers. This article takes a different perspective and explores first the evolutionary interests and leverages of the fungal cultivars, then dissects eight potential evolutionary conflicts between ants and cultivars. Three types of ant-cultivar conflict are examined in depth. First, ant-cultivar conflict over the ant sex ratio is predicted because the cultivars are dispersed by female foundresses but not by males; cultivars thus may be selected to bias the ant sex ratio toward females. Second, ant-cultivar conflict over fungal sexual reproduction exists if the fungi are able to escape from the symbiosis and live independently, as is implied by phylogenetic analyses of the fungi; this conflict is exacerbated in colonies that experience queen death or senescence. A literature review reveals that sexual fruiting of attine cultivars is more common than has been traditionally realized and often occurs in moribund colonies. Third, the routine transplanting of fungal mycelium by ants could generate, through sensory-biased symbiont choice, selection favoring fungal features that increase the likelihood of transplantation within nests (symbiont drive) but that are detrimental to the survival of the whole colony. A balanced perspective incorporating both ant and fungal interests emerges as a more appropriate framework than the traditional myrmicocentric perspective. Indeed, the attine symbiosis offers unique experimental opportunities (cultivar switch experiments) to unravel the evolutionary dynamics of conflict and cooperation between ant and fungal partners.     

A Brazilian Population of the Asexual Fungus-Growing Ant Mycocepurus smithii (Formicidae,Myrmicinae, Attini) Cultivates Fungal Symbionts with Gongylidia-Like Structures

Attine ants cultivate fungi as their most important food source and in turn the fungus is nourished, protected against harmful microorganisms, and dispersed by the ants. This symbiosis evolved approximately 50–60 million years ago in the late Paleocene or early Eocene, and since its origin attine ants have acquired a variety of fungal mutualists in the Leucocoprineae and the distantly related Pterulaceae. The most specialized symbiotic interaction is referred to as “higher agriculture” and includes leafcutter ant agriculture in which the ants cultivate the single species Leucoagaricus gongylophorus. Higher agriculture fungal cultivars are characterized by specialized hyphal tip swellings, so-called gongylidia, which are considered a unique, derived morphological adaptation of higher attine fungi thought to be absent in lower attine fungi. Rare reports of gongylidia-like structures in fungus gardens of lower attines exist, but it was never tested whether these represent rare switches of lower attines to L. gonglyphorus cultivars or whether lower attine cultivars occasionally produce gongylidia. Here we describe the occurrence of gongylidia-like structures in fungus gardens of the asexual lower attine ant Mycocepurus smithii. To test whether M. smithii cultivates leafcutter ant fungi or whether lower attine cultivars produce gongylidia, we identified the M. smithii fungus utilizing molecular and morphological methods. Results shows that the gongylidia-like structures of M. smithii gardens are morphologically similar to gongylidia of higher attine fungus gardens and can only be distinguished by their slightly smaller size. A molecular phylogenetic analysis of the fungal ITS sequence indicates that the gongylidia-bearing M. smithii cultivar belongs to the so-called “Clade 1”of lower Attini cultivars. Given that M. smithii is capable of cultivating a morphologically and genetically diverse array of fungal symbionts, we discuss whether asexuality of the ant host maybe correlated with low partner fidelity and active symbiont choice between fungus and ant mutualists.     

Escovopsioides nivea is a non-specific antagonistic symbiont of ant-fungal crops

Fungus-growing attine ants maintain a mutualistic relationship with basidiomycete fungi which they cultivate for food. In addition to the fungal partner, attine ant colonies harbor a myriad of microorganisms, including the genus Escovopsis, fungal parasites of the ant crops. Because Escovopsioides nivea is phylogenetically close to Escovopsis, previous studies assumed it has a negative interaction in the ant-fungus association. Here, we present an extended phylogeny of E. nivea based on new collections from different attine ant genera found in different localities. We also carried out co-culture experiments between E. nivea with different fungal cultivars. Our results suggest E. nivea is a symbiont of attine ant colonies, which inhibits the growth of fungal crops, supporting the hypothesis it is antagonistic to the system. However, the patterns of interaction between E. nivea and fungal crops differ from those shown by Escovopsis, suggesting a different evolution from that of the parasite.     

Evolution of ant-cultivar specialization and cultivar switching in Apterostigma fungus-growing ants

Almost all of the more than 200 species of fungus-growing ants (Formicidae: Attini) cultivate litter-decomposing fungi in the family Lepiotaceae (Basidiomycota: Agaricales). The single exception to this rule is a subgroup of ant species within the lower attine genus Apterostigma, which cultivate pterulaceous fungi distantly related to the Lepiotaceae. Comparison of cultivar and ant phylogenies suggests that a switch from lepiotaceous to pterulaceous fungiculture occurred only once in the history of the fungus-growing ants. This unique switch occurred after the origin of the genus Apterostigma, such that the basal Apterostigma lineages retained the ancestral attine condition of lepiotaceous fungiculture, and none of the Apterostigma lineages in the monophyletic group of pterulaceous fungiculturists are known to have reverted back to lepiotaceous fungiculture. The origin of pterulaceous fungiculture in attine ants may have involved a unique transition from the ancestral cultivation of litter-decomposing lepiotaceous fungi to the cultivation of wood-decomposing pterulaceous fungi. Phylogenetic analyses further indicate that distantly related Apterostigma ant species sometimes cultivate the same cultivar lineage, indicating evolutionarily frequent, and possibly ongoing, exchanges of fungal cultivars between Apterostigma ant species. The pterulaceous cultivars form two sister clades, and different Apterostigma ant lineages are invariably associated with, and thus specialized on, only one of the two cultivar clades. However, within clades Apterostigma ant species are able to switch between fungi. This pattern of broad specialization by attine ants on defined cultivar clades, coupled with flexible switching between fungi within cultivar clades, is also found in other attine lineages and appears to be a general phenomenon of fungicultural evolution in all fungus-growing ants.     

A preference assay for quantifying symbiont choice in fungus-growing ants (Attini, Formicidae)

We describe a bioassay for the quantification of cultivar preference (symbiont choice) of fungus-growing ants. The bioassay simultaneously presents mycelium of multiple pure cultivar genotypes to worker ants in a cafeteria-style test arena, and preferred versus non-preferred cultivar genotypes can then be identified based on the ants’ quantifiable behavioral tendencies to convert any of the offered mycelium into a fungus garden. Under natural conditions, fungus-growing ants are likely to express such cultivar preferences when mutant cultivars arise in a garden, or when colonies acquire a novel cultivar from a neighboring colony to replace their resident cultivar. We show that workers from different nests of the fungus-growing ant Cyphomyrmex costatus exhibit repeatable preferences vis-à-vis specific cultivar genotypes. The identified preferred and rejected cultivars can then be used in a performance assay to test whether the ants prefer cultivar genotypes that are superior in enhancing colony fitness (measured, for example, as garden productivity or colony growth), as predicted by symbiont-choice theory. Received 24 February 2006; revised 23 June 2006; accepted 26 June 2006.     

Pathogenicity of Escovopsis weberi: The parasite of the attine ant-microbe symbiosis directly consumes the ant-cultivated fungus

Fungi in the genus Escovopsis are known only from the fungus gardens of attine ants. Previous work has established that these anamorphic fungi, allied with the Hypocreales, are specialized and potentially virulent parasites of the ancient mutualism between attine ants and their fungal cultivars. It is unclear whether the primary nutrient source for the pathogen is the mutualist fungal cultivar or the vegetative substrate placed on the gardens by the ants. Here, we determine whether Escovopsis weberi is a parasite of the fungal cultivar, a competitor for the leaf substrate, or both. Bioassays reveal that E. weberi exhibits rapid growth on pure cultivar and negligible growth on sterilized leaf fragments. Light microscopy examination of hyphalhyphal interactions between E. weberi and the ant fungal cultivar indicate that E. weberi, unlike invasive necrotrophs that always penetrate host hyphae, can secrete compounds that break down host mycelium before contact occurs. Thus, E. weberi is a necrotrophic parasite of the fungal cultivar of attine ants.     

Microfungal “Weeds” in the Leafcutter Ant Symbiosis

Leafcutter ants (Formicidae: tribe Attini) are well-known insects that cultivate basidiomycete fungi (Agaricales: Lepiotaceae) as their principal food. Fungus gardens are monocultures of a single cultivar strain, but they also harbor a diverse assemblage of additional microbes with largely unknown roles in the symbiosis. Cultivar-attacking microfungi in the genus Escovopsis are specialized parasites found only in association with attine gardens. Evolutionary theory predicts that the low genetic diversity in monocultures should render ant gardens susceptible to a wide range of diseases, and additional parasites with roles similar to that of Escovopsis are expected to exist. We profiled the diversity of cultivable microfungi found in 37 nests from ten Acromyrmex species from Southern Brazil and compared this diversity to published surveys. Our study revealed a total of 85 microfungal strains. Fusarium oxysporum and Escovopsis were the predominant species in the surveyed gardens, infecting 40.5% and 27% of the nests, respectively. No specific relationship existed regarding microfungal species and ant-host species, ant substrate preference (dicot versus grass) or nesting habit. Molecular data indicated high genetic diversity among Escovopsis isolates. In contrast to the garden parasite, F. oxysporum strains are not specific parasites of the cultivated fungus because strains isolated from attine gardens have similar counterparts found in the environment. Overall, the survey indicates that saprophytic microfungi are prevalent in South American leafcutter ants. We discuss the antagonistic potential of these microorganisms as “weeds” in the ant–fungus symbiosis.     

Antagonistic bacterial interactions help shape host-symbiont dynamics within the fungus-growing ant-microbe mutualism

Conflict within mutually beneficial associations is predicted to destabilize relationships, and theoretical and empirical work exploring this has provided significant insight into the dynamics of cooperative interactions. Within mutualistic associations, the expression and regulation of conflict is likely more complex than in intraspecific cooperative relationship, because of the potential presence of: i) multiple genotypes of microbial species associated with individual hosts, ii) multiple species of symbiotic lineages forming cooperative partner pairings, and iii) additional symbiont lineages. Here we explore complexity of conflict expression within the ancient and coevolved mutualistic association between attine ants, their fungal cultivar, and actinomycetous bacteria (Pseudonocardia). Specifically, we examine conflict between the ants and their Pseudonocardia symbionts maintained to derive antibiotics against parasitic microfungi (Escovopsis) infecting the ants' fungus garden. Symbiont assays pairing isolates of Pseudonocardia spp. associated with fungus-growing ants spanning the phylogenetic diversity of the mutualism revealed that antagonism between strains is common. In contrast, antagonism was substantially less common between more closely related bacteria associated with Acromyrmex leaf-cutting ants. In both experiments, the observed variation in antagonism across pairings was primarily due to the inhibitory capabilities and susceptibility of individual strains, but also the phylogenetic relationships between the ant host of the symbionts, as well as the pair-wise genetic distances between strains. The presence of antagonism throughout the phylogenetic diversity of Pseudonocardia symbionts indicates that these reactions likely have shaped the symbiosis from its origin. Antagonism is expected to prevent novel strains from invading colonies, enforcing single-strain rearing within individual ant colonies. While this may align ant-actinomycete interests in the bipartite association, the presence of single strains of Pseudonocardia within colonies may not be in the best interest of the ants, because increasing the diversity of bacteria, and thereby antibiotic diversity, would help the ant-fungus mutualism deal with the specialized parasites.     

Phylogenetic patterns of ant–fungus associations indicate that farming strategies,not only a superior fungal cultivar,explain the ecological success of leafcutter ants

To elucidate fungicultural specializations contributing to ecological dominance of leafcutter ants, we estimate the phylogeny of fungi cultivated by fungus‐growing (attine) ants, including fungal cultivars from (i) the entire leafcutter range from southern South America to southern North America, (ii) all higher‐attine ant lineages (leafcutting genera Atta, Acromyrmex; nonleafcutting genera Trachymyrmex, Sericomyrmex) and (iii) all lower‐attine lineages. Higher‐attine fungi form two clades, Clade‐A fungi (Leucocoprinus gongylophorus, formerly Attamyces) previously thought to be cultivated only by leafcutter ants, and a sister clade, Clade‐B fungi, previously thought to be cultivated only by Trachymyrmex and Sericomyrmex ants. Contradicting this traditional view, we find that (i) leafcutter ants are not specialized to cultivate only Clade‐A fungi because some leafcutter species ranging across South America cultivate Clade‐B fungi; (ii) Trachymyrmex ants are not specialized to cultivate only Clade‐B fungi because some Trachymyrmex species cultivate Clade‐A fungi and other Trachymyrmex species cultivate fungi known so far only from lower‐attine ants; (iii) in some locations, single higher‐attine ant species or closely related cryptic species cultivate both Clade‐A and Clade‐B fungi; and (iv) ant–fungus co‐evolution among higher‐attine mutualisms is therefore less specialized than previously thought. Sympatric leafcutter ants can be ecologically dominant when cultivating either Clade‐A or Clade‐B fungi, sustaining with either cultivar‐type huge nests that command large foraging territories; conversely, sympatric Trachymyrmex ants cultivating either Clade‐A or Clade‐B fungi can be locally abundant without achieving the ecological dominance of leafcutter ants. Ecological dominance of leafcutter ants therefore does not depend primarily on specialized fungiculture of L. gongylophorus (Clade‐A), but must derive from ant–fungus synergisms and unique ant adaptations.     

Extensive exchange of fungal cultivars between sympatric species of fungus-growing ants

Fungal cultivars of fungus-growing ants (Attini, Formicidae) are carried by dispersing queens from parent to offspring nest. This vertical cultivar transmission between generations is thought to result in long-term ant-fungus coevolution and selection for beneficial cultivar traits that maximize harvests and thus colony productivity. In contrast to this traditional view of vertical cultivar transmission, frequent horizontal cultivar transmission between ant species is implicated by a phylogenetic analysis of 72 cultivars propagated by two fungus-growing ant species coexisting sympatrically in central Panama. The two ant species are specialized on the same group of closely related cultivars, but in six of 12 cultivar clades identifiable within this group, cultivars from both ant species were united in the same clade. Five of these 'mixed' clades were supported by bootstrap values of about 90% or higher. In one instance, colonies from the two ant species cultivated the same, genetically identical, cultivar clone. These phylogenetic patterns indicate that: (i) cultivar exchanges between the two ant species occur routinely throughout ecological time; and that (ii) coevolutionary processes between ants and their fungi are more diffuse than previously assumed. Because the two ant species are specialized on a narrow group of closely related cultivars that they regularly exchange among each other, but not with other sympatric ant species, cultivar exchanges are constrained, most likely, by ant preferences for their own cultivar group or by stringent selection against transitions of ant lineages to distantly related cultivars.     

Behavioral ecology and natural history of Blepharidatta brasiliensis (Formicidae, Blepharidattini)

Fungus-growing ants (Attini, Formicidae) originated about 45–65 million years ago when forging a mutualistic association with basidiomycete fungi (Lepiotaceae). Here we use information on the biology of the non-leafcutting fungus-growing ants and their close relatives in the genus Blepharidatta to evaluate hypotheses for the evolutionary origin of fungus-growing behavior in attine ants. Observations on the natural history, ecology, and behavior of the Amazonian species Blepharidatta brasiliensis are reported here for the first time. Like most attine species, B. brasiliensis and the great majority of species in the tribe Blepharidattini are inhabitants of moist tropical rainforest, suggesting a rainforest habitat also for the ancestral attine ant. The ancestral attine was probably a leaf litter dweller, building small to medium sized nests (e.g., 20–200 workers) either between leaves in the litter or in decaying wood on the rainforest floor. Received 20 December 2005; revised 1 March 2006; accepted 7 March 2006.     

EVOLUTIONARY TRANSITIONS IN ENZYME ACTIVITY OF ANT FUNGUS GARDENS

Fungus‐growing (attine) ants and their fungal symbionts passed through several evolutionary transitions during their 50 million year old evolutionary history. The basal attine lineages often shifted between two main cultivar clades, whereas the derived higher‐attine lineages maintained an association with a monophyletic clade of specialized symbionts. In conjunction with the transition to specialized symbionts, the ants advanced in colony size and social complexity. Here we provide a comparative study of the functional specialization in extracellular enzyme activities in fungus gardens across the attine phylogeny. We show that, relative to sister clades, gardens of higher‐attine ants have enhanced activity of protein‐digesting enzymes, whereas gardens of leaf‐cutting ants also have increased activity of starch‐digesting enzymes. However, the enzyme activities of lower‐attine fungus gardens are targeted primarily toward partial degradation of plant cell walls, reflecting a plesiomorphic state of nondomesticated fungi. The enzyme profiles of the higher‐attine and leaf‐cutting gardens appear particularly suited to digest fresh plant materials and to access nutrients from live cells without major breakdown of cell walls. The adaptive significance of the lower‐attine symbiont shifts remains unclear. One of these shifts was obligate, but digestive advantages remained ambiguous, whereas the other remained facultative despite providing greater digestive efficiency.     

Coevolution between attine ants and actinomycete bacteria: a reevaluation

We reassess the coevolution between actinomycete bacteria and fungus-gardening (attine) ants. Actinomycete bacteria are of special interest because they are metabolic mutualists of diverse organisms (e.g., in nitrogen-fixation or antibiotic production) and because Pseudonocardia actinomycetes are thought to serve disease-suppressing functions in attine gardens. Phylogenetic information from culture-dependent and culture-independent microbial surveys reveals (1) close affinities between free-living and ant-associated Pseudonocardia, and (2) essentially no topological correspondence between ant and Pseudonocardia phylogenies, indicating frequent bacterial acquisition from environmental sources. Identity of ant-associated Pseudonocardia and isolates from soil and plants implicates these environments as sources from which attine ants acquire Pseudonocardia. Close relatives of Atta leafcutter ants have abundant Pseudonocardia, but Pseudonocardia in Atta is rare and appears at the level of environmental contamination. In contrast, actinomycete bacteria in the genera Mycobacterium and Microbacterium can be readily isolated from gardens and starter-cultures of Atta. The accumulated phylogenetic evidence is inconsistent with prevailing views of specific coevolution between Pseudonocardia, attine ants, and garden diseases. Because of frequent acquisition, current models of Pseudonocardia-disease coevolution now need to be revised. The effectiveness of Pseudonocardia antibiotics may not derive from advantages in the coevolutionary arms race with specialized garden diseases, as currently believed, but from frequent recruitment of effective microbes from environmental sources. Indeed, the exposed integumental structures that support actinomycete growth on attine ants argue for a morphological design facilitating bacterial recruitment. We review the accumulated evidence that attine ants have undergone modifications in association with actinomycete bacteria, but we find insufficient support for the reverse, modifications of the bacteria resulting from the interaction with attine ants. The defining feature of coevolution--reciprocal modification--therefore remains to be established for the attine ant-actinomycete mutualism.     

The origin of the attine ant-fungus mutualism.

Cultivation of fungus for food originated about 45-65 million years ago in the ancestor of fungus-growing ants (Formicidae, tribe Attini), representing an evolutionary transition from the life of a hunter-gatherer of arthropod prey, nectar, and other plant juices, to the life of a farmer subsisting on cultivated fungi. Seven hypotheses have been suggested for the origin of attine fungiculture, each differing with respect to the substrate used by the ancestral attine ants for fungal cultivation. Phylogenetic information on the cultivated fungi, in conjunction with information on the nesting biology of extant attine ants and their presumed closest relatives, reveal that the attine ancestors probably did not encounter their cultivars-to-be in seed stores (von Ihering 1894), in rotting wood (Forel 1902), as mycorrhizae (Garling 1979), on arthropod corpses (von Ihering 1894) or ant faeces in nest middens (Wheeler 1907). Rather, the attine ant-fungus mutualism probably arose from adventitious interactions with fungi that grew on walls of nests built in leaf litter (Emery 1899), or from a system of fungal myrmecochory in which specialized fungi relied on ants for dispersal (Bailey 1920) and in which the ants fortuitously vectored these fungi from parent to offspring nests prior to a true fungicultural stage. Reliance on fungi as a dominant food source has evolved only twice in ants: first in the attine ants, and second in some ant species in the solenopsidine genus Megalomyrmex that either coexist as trophic parasites in gardens of attine hosts or aggressively usurp gardens from them. All other known ant-fungus associations are either adventitious or have nonnutritional functions (e.g., strengthening of carton-walls in ant nests). There exist no unambiguous reports of facultative mycophagy in ants, but such trophic ant-fungus interactions would most likely occur underground or in leaf litter and thus escape easy observation. Indirect evidence of fungivory can be deduced from contents of the ant alimentary canal and particularly from the contents of the infrabuccal pocket, a pharyngeal device that filters out solids before liquids pass into the intestine. Infrabuccal pocket contents reveal that ants routinely ingest fungal spores and hyphal material. Infrabuccal contents are eventually expelled as a pellet on nest middens or away from the nest by foragers, suggesting that the pellet provides fungi with a means for the dispersal of spores and hyphae. Associations between such "buccophilous" fungi and ants may have originated multiple times and may have become elaborated and externalized in the case of the attine ant-fungus mutualism. Thus, contrary to the traditional model in which attine fungi are viewed as passive symbionts that happened to come under ant control, this alternative model of a myrmecochorous origin of the attine mutualism attributes an important role to evolutionary modifications of the fungi that preceded the ant transition from hunter-gatherer to fungus farmer.     

Towards a molecular understanding of symbiont function: Identification of a fungal gene for the degradation of xylan in the fungus gardens of leaf-cutting ants

Background  

Leaf-cutting ants live in symbiosis with a fungus that they rear for food by providing it with live plant material. Until recently the fungus' main inferred function was to make otherwise inaccessible cell wall degradation products available to the ants, but new studies have shed doubt on this idea. To provide evidence for the cell wall degrading capacity of the attine ant symbiont, we designed PCR primers from conserved regions of known xylanase genes, to be used in PCR with genomic DNA from the symbiont as template. We also measured xylanase, cellulase and proteinase activities in the fungus gardens in order to investigate the dynamics of degradation activities.     

Landscape genomics of an obligate mutualism: Concordant and discordant population structures between the leafcutter ant Atta texana and its two main fungal symbiont types

To explore landscape genomics at the range limit of an obligate mutualism, we use genotyping‐by‐sequencing (ddRADseq) to quantify population structure and the effect of host–symbiont interactions between the northernmost fungus‐farming leafcutter ant Atta texana and its two main types of cultivated fungus. Genome‐wide differentiation between ants associated with either of the two fungal types is of the same order of magnitude as differentiation associated with temperature and precipitation across the ant's entire range, suggesting that specific ant–fungus genome–genome combinations may have been favoured by selection. For the ant hosts, we found a broad cline of genetic structure across the range, and a reduction of genetic diversity along the axis of range expansion towards the range margin. This population‐genetic structure was concordant between the ants and one cultivar type (M‐fungi, concordant clines) but discordant for the other cultivar type (T‐fungi). Discordance in population‐genetic structures between ant hosts and a fungal symbiont is surprising because the ant farmers codisperse with their vertically transmitted fungal symbionts. Discordance implies that (a) the fungi disperse also through between‐nest horizontal transfer or other unknown mechanisms, and (b) genetic drift and gene flow can differ in magnitude between each partner and between different ant–fungus combinations. Together, these findings imply that variation in the strength of drift and gene flow experienced by each mutualistic partner affects adaptation to environmental stress at the range margin, and genome–genome interactions between host and symbiont influence adaptive genetic differentiation of the host during range evolution in this obligate mutualism.     

The Fungus-culturing Behavior of Ants

A colony of attine ants begins with a recently fecundated femalecarrying hyphae from the parental garden in a pellet in an infrabuccalpocket. All future food of the colony will be derived from thisnucleus. She digs a cavity in the ground, ejects this pelletand manures it with her liquid excrement. As the hyphae proliferate,eggs are laid on them and the colony is launched. She continuallylicks both the hyphae and the brood. Thus, both salivary andanal excretions play a vital role in the beginning of a colonyand this pattern is repeated by the resulting workers. About60–65% of them in Atta are the minima and these are intimatelyinvolved in brood and fungus care. Their excretions are disproportionatelylarge. About 1/3 of the workers in Atta are 4–6 mm mediaand these cut and prepare the substrate. The 7–9 mm maximasizes and the soldiers (over 9 mm) are less directly involvedin culturing the fungus. The effectiveness of fungus culturing is shown by the rapidbuild-up of gardens. The ants maintain their garden despitesurrounding contamination after a fragment with ants is introducedto a plate of sterile nutrient agar.