Evolution, 55(10), 2001, pp. 1980±1991

PARTIAL INCOMPATIBILITY BETWEEN AND SYMBIOTIC FUNGI IN TWO SYMPATRIC OF LEAF-CUTTING ANTS

A. N. M. BOT,1,2 S. A. REHNER,3,4 AND J. J. BOOMSMA1,5,6 1Department of Ecology and Genetics, Institute of Biological Sciences, University of Aarhus, 8000 Aarhus C, Denmark 3Department of Biology, P.O. Box 23360, University of Puerto Rico, Rio Piedras, Puerto Rico, 00931-3360 5Department of Population Ecology, Zoological Institute, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark

Abstract. We investigate the nature and duration of incompatibility between certain combinations of Acromyrmex leaf-cutting ants and symbiotic fungi, taken from sympatric colonies of the same or a related species. -fungus incompatibility appeared to be largely independent of the ant species involved, but could be explained partly by genetic differences among the fungus cultivars. Following current theoretical considerations, we develop a hypothesis, originally proposed by S. A. Frank, that the observed incompatibilities are ultimately due to competitive interactions between genetically different fungal lineages, and we predict that the ants should have evolved mechanisms to prevent such competition between cultivars within a single garden. This requires that the ants are able to recognize unfamiliar fungi, and we show that this is indeed the case. Ampli®ed fragment length polymorphism genotyping further shows that the two sympatric Acromyrmex species share each other's major lineages of cultivar, con®rming that horizontal transfer does occasionally take place. We argue and provide some evidence that chemical substances produced by the fungus garden may mediate recognition of alien fungi by the ants. We show that incompatibility between ants and transplanted, genetically different cultivars is indeed due to active killing of the novel cultivar by the ants. This incompatibility disappears when ants are force-fed the novel cultivar for about a week, a result that is consistent with our hypothesis of recognition induced by the resident fungus and eventual replacement of incompatibility compounds during force-feeding.

Key words. Attini, compatibility, fungi, fungus-growing ants, mutualism, speci®city, symbiosis.

Received February 5, 2000. Accepted April 11, 2001.

Obligate mutualisms are shaped and maintained by a com- mechanisms (Frank 1998). However, even in the most suc- plex array of selection forces. On one hand, mutually ben- cessful mutualistic partnerships there is no a priori reason e®cial traits are favored as long as they increase the ef®ciency why partial exploitation of one partner by the other would of resource acquisition, metabolic performance, and/or de- not yield even higher ®tness than pure cooperation. This im- fense. On the other hand, sel®sh traits in each partner may plies that mutualistic partners may occasionally pursue their be maintained to some extent as long as they do not jeopardize own sel®sh reproductive interest (e.g., Herre and West 1997) the mutually bene®cial symbiosis. Much of the evolutionary and that the separation between parasitism and mutualism complexity shaping mutualistic symbioses can thus be traced becomes blurred (Bronstein 1994; Thompson 1994; Herre back to Hamiltonian concepts of con¯ict and cooperation 1999; Herre et al. 1999). (Hamilton 1964). Lasting cooperation between independent Obligate symbiosis provides special opportunities and con- genomes belonging to the same breeding population is nor- straints for mutualistic interactions. Symbionts living inside mally maintained only when inclusive ®tness bene®ts via kin or in close association with a single host individual are nor- are signi®cant. In the absence of kinship in interactions be- mally either closely related among each other (when they tween species, competition prevails over cooperation, unless belong to the same asexual clone) or unrelated (when they the kinship incentives are replaced by reciprocity and mutual belong to different lineages that independently colonize a policing (Trivers 1971; Frank 1996b; Leigh 1999). When host). In the former case, the ®tness interests of individual genomes belonging to different gene pools (i.e., taxa) evolve symbionts are largely or completely aligned with the ®tness mutualistic cooperation, they usually obtain a joint niche that of the host (Herre et al. 1999), but this is not so in the latter allows a consistently higher ®tness than either of the single- case. As outlined by Frank (1996a) and previously by Eber- species niches, for example, as in lichens, corals, and my- hard (1980), Cosmides and Tooby (1981), Hoekstra (1987), corrhizal symbioses (Thompson 1994). Although cooperation and Hurst (1994), hosts and symbionts are in con¯ict over across taxa excludes Hamiltonian relatedness in a strict sense, many of the broader conceptual considerations regarding the mixing of symbiont lineages. Symbionts will always be such associations are strongly reminiscent of inclusive ®t- under selection to realize some horizontal transmission to ness, with reciprocity and policing as stability-enhancing reduce competition with close relatives and colonize new hosts (Hamilton and May 1977; Frank 1997). However, the successful establishment of additional unrelated symbionts 2 Present address: Institute of Evolutionary and Ecological Sci- ences, University of Leiden, Kaiserstraat 63, P.O. Box 9516, 2300 in an individual host tends to induce competition between RA Leiden, The Netherlands; E-mail: [email protected]. symbiont lineages. The concomitant reduction in average ®t- 4 Present address: U.S. Department of Agriculture, Agricultural ness of all symbionts within a single host, and thus also of Research Service, Plant Sciences Institute, Control Labora- host ®tness, can be referred to as virulence (Frank 1996a), tory, Building 011A, Room 214, BARC-W, Beltsville, Maryland 20705; E-mail: [email protected]. in parallel with the negative effect of parasites or diseases 6 Corresponding author. E-mail: [email protected]. on the ®tness of their hosts (e.g., Lively 1999). Frank's theory 1980 ᭧ 2001 The Society for the Study of Evolution. All rights reserved. INCOMPATIBILITY BETWEEN ANTS AND FUNGI 1981 of host-symbiont con¯ict over mixing of symbiotic lineages parasite-host interactions, where the host consistently evolves predicts that hosts will evolve effective means to control to minimize the interaction while imposing constant pressure symbiont transmission when a number of speci®c assump- for pathogen specialization (Thompson 1999). It has also tions are met. This theory makes speci®c predictions regard- become clear that not all combinations of hosts and mutu- ing the low genetic variation expected among symbionts alistic symbionts are equally ®t (e.g., Bever 1999 and ref- within a single host, but it does not allow inferences about erences therein). For example, in a recent study on Bradyr- the extent of genetic polymorphism that can be maintained hizobium nitrogen-®xing bacteria and the legume host Am- in entire breeding populations of symbionts. The theory spec- phicarpaea bracteata, Wilkinson et al. (1996) showed that i®es that conditions for invasion of host modi®er genes that all Bradyrhizobium strains enhance host ®tness. However, the restrict mixing of symbiont lineages are more lenient when locally coexisting combinations performed better than the frequency-dependent selection maintains high levels of sym- allopatric combinations, and the performance of the latter biont polymorphism in the population (Frank 1996a). How- was related to the genetic distance from the experimental ever, there are few speci®c models (Law 1985; Bever 1999; host to the native host. Parker 1999) and few empirical data (Rowan and Powers Here we present an experimental study addressing ques- 1991; Martin 1992; Wilkinson et al. 1996; Parker 1999) ad- tions of fungus cultivar diversity within and across their Ac- dressing issues of populationwide polymorphism of mutu- romyrmex leaf-cutting ant hosts. Using reciprocal transplant alistic symbionts. experiments, we investigate the speci®city, short-term per- There may be a good reason for the absence of theoretical formance, and compatibility between fungus cultivars and predictions for symbiont polymorphism. Negative frequency- ant colonies of two closely related sympatric species, A. oc- dependent selection is of signi®cant importance for main- tospinosus and A. echinatior. We combine the results of these taining high degrees of polymorphism in parasite-host inter- transplant experiments with ampli®ed fragment length poly- actions (Hamilton 1980; Seger 1988, 1992; Ebert and Ham- morphism (AFLP) data estimating genetic variation of the ilton 1996). A corollary of this hypothesis, that parasites are entire population of fungus cultivars. We analyze and review better adapted to and thus more successful with their local some of the mechanisms responsible for partial incompati- hosts than with hosts from other populations, has recently bility and interpret our results in the light of Frank's (1996a) received considerable support from empirical studies (Lively theoretical concepts on host-symbiont con¯ict over the mix- 1989, 1999; Lively and Dybdahl 2000; Ebert 1994; for a ing of symbiotic lineages. review see Kaltz and Shykoff 1998). At the other extreme, pure mutualistic symbioses without any sel®sh tendencies Natural History of the Model System have been hypothesized to be shaped by positive frequency- dependent selection favoring a single most productive lineage Attine ants cultivate fungus cultivars derived from ®ve of mutualistic symbiont (Law 1985; Law and Koptur 1986). different lineages of mushrooms, four from within the Le- This implies that novel alleles arising in symbionts do not piotaceae and one from within the Tricholomataceae. Al- necessarily have an advantage and that, even in metapopu- though the phylogenies of the ants and their fungus cultivars lations, local dynamics and turnover should be less erratic are congruent at the deepest levels, coevolutionary patterns than in antagonistic interactions, so that polymorphism between ants and their preferred cultivars are reticulate due should remain relatively low (Parker 1999). However, our to lateral transfer of cultivars and speci®city across species current understanding of mutualistic interactions as arrange- is relatively low (Mueller et al. 1998; S. A. Rehner, U. G. ments for mutual net bene®ts despite reciprocal exploitation Mueller, C. R. Currie, and T. M. Schultz, unpubl. ms.). The (Frank 1996a; Herre 1999) addresses intermediate situations fungus cultivars of the evolutionary advanced leaf-cutting where both positive frequency-dependence and negative fre- ants (the genera Atta and Acromyrmex) are derived from the quency-dependence are likely to apply, consistent with the Leucocoprineae (Lepiotaceae; Johnson 1999) and no closely modeling approach by Bever (1999). In such a conceptual related free-living species have yet been discovered, despite framework, mutualisms are shaped by evolutionary arms rac- intensive sampling (Chapela et al. 1994). In contrast to the es between symbionts with variable tendencies of sel®shness lower attines (see below), the symbiotic fungi of the higher (virulence) and hosts with variable capacities to control sym- attines have undergone both morphological and physiological biont transmission. The balance between positive and neg- adaptations in their association with ants (Weber 1966, 1972; ative frequency dependence may thus differ from case to case, Mueller et al. 2001), which explains why options for living depending on speci®c biological properties and constraints outside the mutualism are severely restricted, if not com- of partners. pletely absent. The ants are likewise fully dependent on their The issue of extant genetic variation in symbiont lineages fungus cultivar for food. is further complicated by geographic variation between pop- In contrast to mycorrhizae (e.g., Bever et al. 1996; Sanders ulations of mutualists (Thompson 1994, 1999). At increasing and Fitter 1992) and the legume-Bradyrhizobium mutualism geographical scales, populations may both be subjected to (Wilkinson et al. 1996), where horizontal transmission is the genetic drift of symbionts and they may adaptively converge rule, the fungal mutualist of leaf-cutting ants is vertically on gradients of different mutualistic partners resulting in sta- transmitted from one ant generation to the next (Weber 1972). ble geographic mosaics (Parker 1999; Thompson 1999). Al- This happens when gynes (winged, prospective queens) take though ®rm generalizations are as yet impossible, there seems a small fragment of the fungus garden from their natal colony to be an emerging consensus that local coadaptation between along on their mating ¯ight and use this to start their own mutualistic symbionts is unlikely as pronounced as in certain fungus garden. Lateral transfers may occur, however, after 1982 A. N. M. BOT ET AL. the loss of a fungus garden (Adams et al. 2001) or during bene®ts from acquiring a potentially superior cultivar is mi- the colony founding stage. Opportunities of the latter type nor in comparison to the short-term risk of suffering im- may occur when colonies are initiated by multiple foundress- mediate ®tness loss or even death of the fungus garden due es, as is the case in some Acromyrmex and Atta species (Riss- to competition between the resident and the introduced cul- ing et al. 1989; Mintzer 1990; possibly also in one of our tivar. study species A. echinatior, Bekkevold et al. 1999). Alter- Both the ants and their resident fungus cultivar are thus natively (but not mutually exclusive), transfers of cultivars expected to express hostile reactions to incoming unrelated may be facilitated by close proximity of potentially com- strains of fungus, but to accept fungal strains from other peting founding colonies (Autuori 1950; Rissing et al. 1989), colonies when they are genetically identical to the resident especially when they aggregate under the same log or stone cultivar at speci®c recognition or compatibility loci. In the (Rissing et al. 1989; observed for our other study species, A. absence of knowledge on the identity of these compatibility octospinosus, in Panama, U. G. Mueller, pers. comm.). loci, genetic similarity can be estimated by techniques that In the terminology used by Frank (1996a), the natural his- measure genomewide variation at mostly neutral loci. The tory of the symbiosis between attine ants and their basidio- latter type of variation can be used as a proxy for between- mycete fungus can be characterized as follows. The symbiosis strain variation at incompatibility loci, although considerable is an obligate mutualism and has a clear net bene®cial effect noise on this predictor variable cannot be excluded (see Dis- on both partners. Dikaryotic mycelia of free-living basidio- cussion). mycetes are known for their individualistic behaviors re- Vertical transmission allows the ant hosts to control the sulting from incompatibility reactions among nonrelated movement and mixing of cultivars during mating ¯ights, but strains (see Discussion). The fungus cultivars of the lower this does not preclude confrontations with horizontally trans- attine ants have recently been shown to be closely related to mitted, alien strains later on (Autuori 1950). Cultivars, in (and sometimes indistinguishable from) free-living fungi contrast, have little power to oppose the interests of their (Mueller et al. 1998). This implies that incompatibility mech- hosts, except perhaps for rare sporulation events with the anisms and the potential for sexual reproduction probably potential of horizontal infection of competing strains estab- have not been lost. It is therefore no surprise that vertical lished as fungus gardens of other colonies. The effective transmission of cultivars is already present in the lower at- control by the host ants over cultivar transmission may have tines and possibly represents a trait that predated the origin signi®cantly reduced the populationwide polymorphism for of fungus rearing in the attine tribe (Mueller et al. 2001). incompatibility genes (cf. Douglas 1994; Frank 1996a; see Even the mutualistic fungi of the higher attines are known also Herre et al. 1999). However, it is unlikely to have elim- to fruit on occasions (Muchovej et al. 1991; DoÈrfelt and inated genetic variation altogether, because control is not Creutzburg 1994; Fisher et al. 1994 and references therein). perfect and Acromyrmex populations are subdivided to at least This form of sexual reproduction sometimes occurs after hor- a certain extent, so that local adaptations and population dif- izontal transmission of, or contamination with, alien fungi. ferentiation due to genetic drift are likely to occur (Parker It is usually accompanied by severe worker mortality, often 1999; Thompson 1999). (but not always) followed by the complete collapse of the fungus garden and the death of the colony (DoÈrfelt and MATERIALS AND METHODS Creutzburg 1994; Fisher et al. 1994; A. N. M. Bot, pers. The Fungus Exchange Experiment obs.). This implies that the attine fungus cultivars may have retained competitive traits that enhance the reproduction and/ Three colonies of A. octospinosus (nos. 31, 38, 41) and or transmission of cultivar lineages when mixing of lineages three colonies of A. echinatior (33, 47, 48) were used. These occurs. Because competition between cultivars leads to a re- queenright colonies were part of a larger sample collected in duction of the ®tness of the ants, competitive traits of the January and February 1996 from a remnant of secondary fungus can be considered in the same light as virulence traits growth forest in Gamboa, Republic of Panama. The total area in parasitic symbioses. of the collection site was about 0.3 km2. Within the area the It seems reasonable to assume that the ant hosts are able typical ranges of the two species overlap, although A. octos- to recognize alien cultivars in the same way as they are able pinosus tends to be restricted to shaded habitats within the to recognize and weed out pathogens (Currie et al. 1999a; forest, whereas A. echinatior is more common in partly sun- Currie and Stewart 2001; A. G. Hart, A. N. M. Bot, and M. exposed habitats. The mature colonies sampled were quite J. Brown, unpubl. ms.). The resident fungus should also have spaced out and never within foraging distance of each other. retained mechanisms to recognize alien strains of fungus. For each colony, part of the fungus garden, worker ants, and However, instead of investing considerable resources in the the queen were transported to Denmark and set up as labo- expression of incompatibility virulence, the derived fungal ratory colonies in a climate room with a constant temperature lineages of the higher attines may suf®ce with producing of 25ЊC and a relative humidity of 60±70%. Acromyrmex signaling compounds that characterize a fungal clone and echinatior used to be known as a local form of A. octospi- induce the ants to recognize and remove genetically different nosus, but was recently elevated to full species status on the clones. With or without the help of their resident fungus, the basis of morphological and allozyme evidence (Schultz et al. ants are expected to have evolved ef®cient control mecha- 1998). The Acromyrmex colonies were maintained on a diet nisms to retain their resident fungus in a genetically homo- of bramble leaves (Rubus sp.; cf. Bot and Boomsma 1996) geneous state. This is because, as long as they have a well- and had maintained a stable fungus garden size of circa 1 L functioning cultivar, the chance that they will reap long-term for at least two months prior to the start of the experiment. INCOMPATIBILITY BETWEEN ANTS AND FUNGI 1983

The experimental set-up consisted of small round plastic of cultivars, which contains the six colonies that were used vials (diameter 2.5 cm, height 4 cm) with moist tissue paper for the experiments and is nested within a larger tree (not at the bottom to provide humidity. A fresh green bramble shown) that also includes the cultivars of three Panamanian leaf was folded into the vial and a fungus fragment of ap- species of leaf-cutting ants classi®ed in the Atta (S. A. proximately 80 mg was placed on top of the leaf. The fungus Rehner, U. G. Mueller, C. R. Currie, and T. M. Schultz, fragments used were always collected from the top of the unpubl. ms.). The overall phylogenetic relationships of the fungus garden, because active mycelial growth is restricted fungi were not known at the time when the exchange ex- to this upper region, which therefore tends to be more vig- periments were performed. orous and homogeneous than the older, lower part of the fungus garden. By choosing fungus from the upper part of Experiments on the Background of Incompatibility the garden, any differences in fungus quality between rep- licates was minimized. One pupa was added to each vial, to Two additional experiments were performed to investigate minimize the possibility of abnormal worker behavior re- whether the ant-fungus incompatibility observed in one of sulting from the absence of brood. The experimental sub- the reciprocal transplants was due to antagonistic behavior colonies were completed by adding a total of eight worker of the ants or their resident fungus. Attine ants are known ants (four minors, two media, and two majors) per vial. to transfer fungal enzymes acquired from eating mycelium Fungus garden fragments were combined with ants from and gongylidia in the older (lower) parts of the fungus garden either their original colony or from one of the ®ve other to the freshly incorporated vegetable material and newly colonies, so that all pairwise combinations of ants and fungus grown fungus in the upper part of the garden (Boyd and were tested with two replicates each. The fungus was weighed Martin 1975; Martin et al. 1975). These enzymes are essential on days 0, 2, and 4 of the experiment, and on day 2 the for the predigestion of leaf material. They are ingested by subcolonies were given clean vials with fresh leaves. The the ants and are subsequently deposited as fecal droplets on experiment was repeated six times over a period of ®ve weeks freshly harvested leaf material, so that new inoculates of the in January and February 1998, so that a total of 12 replicates fungus cultivar will be able to grow faster (Boyd and Martin per treatment were available for analysis. In the entire series 1975; Martin et al. 1975). If the cultivar fungi produce chem- of experiments, only 12 of 432 (3%) pupa died and only 19 ical compounds that inhibit the growth of alien strains, it of 3456 (0.5%) workers died. There was never more than one seems not impossible that these compounds could also hitch- mortality case per vial, so that these low mortality rates were hike through the ant gut together with the growth-enhancing assumed not to have affected the condition of fungus frag- enzymes, because: they can then be produced in the central ments. part of the garden, which is most likely to be genetically pure; they will be targeted in the best possible way, because the ants will deposit fecal droplets on any piece of newly Genetic Comparison of Fungus Clones inoculated fungus at the top of the garden that has not yet Genetic relatedness among fungus clones originating from been recognized and rejected as alien; and they only need to different experimental colonies was inferred from analysis of produce relatively subtle incompatibility reactions with frag- AFLP markers. Fungus cultivars from 40 colonies of A. oc- ments of alien fungus, as long as these reactions are suf®cient tospinosus and A. echinatior, collected from the Gamboa pop- to inform the tending ants that some fragments are genetically ulation between 1993 and 1998, were isolated into pure cul- different from the resident fungus cultivar. ture. Isolation was accomplished by transferring small frag- First, we have tested the effect of ant fecal droplets on ments of fungus garden onto petri dishes containing potato isolated, axenic fungus in a reciprocal-cross experiment in- dextrose agar (PDA; Difco, Detroit, MI) adjusted to 50 ␮g/ volving the two most incompatible colonies, A. echinatior ml streptomycin sulfate and penicillin G. Cultivar outgrowths colony 48 and A. octospinosus colony 38, which have ge- from the garden explants were serially subcultured to fresh netically distinct fungus cultivars (see Results). Fecal drop- plates until they were free of contaminant fungi and bacteria. lets of the ants were obtained by holding individual ant work- Mycelium for DNA extractions was produced by transferring ers by the head. This stressful posture usually induced release cultivar strains into liquid potato broth (PDB; Difco) and of a fecal droplet, which could then be collected in a capillary shaking at 100 rpm for approximately 20 days. DNA ex- and applied directly on small tufts of fungus that had been tractions followed Rehner and Samuels (1994), and AFLP subcultured onto fresh medium (PDA, Difco) several days in reactions and data acquisition were performed as described advance. Each of the four combinations of fungus and fecal by Vos et al. (1995). AFLP data were generated for two droplets was replicated eight times. After 24 h, we assessed selective primer combinations, Eco-CC ϫ Mse CAT and Eco- whether the fungus had absorbed the fecal droplets. CC ϫ Mse-CAC. AFLP bands were treated as binary char- Second, we tested whether the incompatibility between acters and scored as present or absent for each cultivar strain ants and fungi was transient. If fungal incompatibility sub- and entered into a data matrix. The AFLP data were analyzed stances are transferred through the gut of ant workers, pro- under the parsimony criterion with PAUP* (Swofford 1998). longed enforced exposure to a novel fungus should make the Phylogenetic trees were inferred from the AFLP data using incompatibility effect gradually disappear, as the ants acquire a heuristic search with random input order and TBR branch enzymes from the new fungus. This could imply that this swapping. Node support was assessed by 2500 bootstrap proximate mechanism is in fact adaptive. Although the ants pseudoreplicates using input and swapping parameters. The are likely to have a direct ®tness interest (sensu Frank 1996a) tree presented in this paper represents a monophyletic group in a genetically homogenous fungus garden, this does not 1984 A. N. M. BOT ET AL. imply that their hostile responses against alien strains of fun- were distinct) were included in the model as main effects, gus should last forever. Antagonistic reactions during the ®rst and colonies were nested within species (random effects). few days are probably quite suf®cient to cull any small quan- The full model also included interaction terms between these tity of introduced alien cultivar. However, retaining this be- variables. Statistical tests were based on Type III expected havior inde®nitely may not be selected for, because it will mean squares when random effects were included in the mod- preclude rare opportunities of obtaining a new fungus garden el, otherwise on Type III sums of squares. The residuals were by raiding a neighboring colony after an irreversible loss of tested for normality by means of Shapiro-Wilk's test (W) the resident fungus garden. The expected parallel interests of using PROC UNIVARIATE in SAS, and variance homosce- both mutualistic partners in a gradual decrease of incompat- dasticity was assessed visually from plotting the residuals ibility between ants and alien fungi may thus imply that the against the predicted values. concentration of compounds of the old fungus in the gut of We used correspondence analysis in JMP (SAS Inst. 1995) the ants serves as the proximate cue to regulate the extent of to quantify the degree of similarity in the pattern of mortality behavioral aggression against alien fungi. between the transplanted fungus cultivars of the six Acro- The persistence of incompatibility was analyzed in a re- myrmex colonies and pooled the data of all 12 replicates also ciprocal-cross experiment with the two most incompatible for this analysis. The obtained correspondence scores were colonies, 38 and 48. For this experiment we used somewhat subsequently compared with the phylogenetic distance of the larger subcolonies, in which the fungus was replaced every fungus cultivars. Correspondence analysis is similar to prin- day by fresh fungus from the source colony of the transplant. cipal component analysis, but uses binary data, which may The treatments and controls had three replicates, each con- be, but are not necessarily, symmetrical. Our hypothesis that sisting of 40 ants (20 minors and 20 medium/large workers), incompatibility is ultimately due to fungus-fungus interac- four pupae, and 0.5 g of fungus. Each subcolony was main- tions and/or to selection on ants to cull novel fragments of tained in a dish of 6-cm diameter, partly covered by a lid, cultivar predicts that patterns of incompatibility should be which was placed inside a nest box of 11 cm by 17 cm. A symmetrical. This means that any degree of incompatibility moist cotton plug was added to each dish to maintain high of ants from colony A toward fungus from colony B should humidity, and bramble leaves were supplied in the nest box be expressed to the same extent as the reciprocal incompat- outside the dish as forage for the ants. The weight of each ibility between ants from colony B and fungus from colony fungus fragment was recorded just before transplanation and A. Correspondence analysis provides a quantitative analysis when replacing it each day, so that the extent of incompat- of the (a)symmetry of interactions by producing two plots, ibility could be inferred from the differences in weight from one describing the fungus-ant (in)compatibility and another one day to the next. This experiment also tested whether the describing the ant-fungus (in)compatibility. Any difference ants were able to recognize new fungus cultivars and whether between these two plots thus indicates asymmetry of the ob- they expressed direct hostile behavior toward new fungus served incompatibility interactions. This analysis incorpo- cultivars. To this end, the behavior of the ants was observed rates both the direct incompatibility between two colonies for about 15 min directly after introducing a fresh fragment and the similarity in incompatibility patterns toward other of fungus. colonies.

Statistical Analysis RESULTS Because not all replicates in the fungus exchange exper- Variable Compatibility between Ants and Fungi iment were set up on the same day, we ®rst tested for the None of the controls where ants tended their own fungus effect of starting date on fungus mortality. We used a ␹2- showed any fungus mortality. Both in these controls and in analysis to test the observed distribution of fungus mortality a number of transplants involving different colonies, the fun- against the expected distribution for each of the 16 (of 36) gal fragments maintained an almost constant weight over the series of ant-fungus replicates where any fungus mortality four days of the experiment (see Fig. 1), with a slight increase took place. The null hypothesis was that mortality was spread in variance over time. However, in other combinations in- evenly over all replicates, and the alternative hypothesis was volving different colonies, fungal fragments died at varying that mortality occurred more or less often within replicates rates, and in some combinations most or all of the 12 replicate that were set up on the same day. The relative changes in fungal fragments died. The 16 P-values from the ␹2-tests for fungal weight from day 0 to day 4 (see Fig. 1) were analyzed independence of fungus mortality of starting date of the ex- by means of a two-way mixed analysis of variance (ANOVA) periment ranged from 0.049 to 0.998. Only one of these P- using PROC GLM in SAS for Windows (ver. 6.12, SAS values was signi®cant at the 5% level prior to a Bonferroni Institute 1994). To achieve normally distributed residuals adjustment, so that we decided to ignore the effect of starting with homogenous variance, the values of the dependent var- date and to pool all data in the subsequent analyses. iable (y ϭ W/W0 ϭ [W4 Ϫ W0]/W0, where W0 and W4 are Figure 1 shows that if the ants of one colony were incom- fungal weights at the beginning and at the end of the exper- patible with the fungus of another colony, the reverse was iment respectively) were subjected to a square root transfor- also the case. Acromyrmex octospinosus colony 38 is the main 1/2 mation: y* ϭ [(W4 Ϫ W0)/W0] if W4 Ͼ W0 and y* ϭϪ[(W0 source of fungus mortality. It is incompatible with all other 1/2 Ϫ W4)/W0] if W0 Ͼ W4. Ant species and fungus ``species'' colonies, irrespective of the species to which they belong. (``species'' for fungus because it was unknown at the time Acromyrmex octospinosus colony 31, on the other hand is of the experiment whether cultivars from the two ant species compatible with two other colonies of the same species and INCOMPATIBILITY BETWEEN ANTS AND FUNGI 1985

FIG. 1. The fungus weight measurements at days 0, 2, and 4 for 12 replicates in the different combinations of ants and fungus from three colonies of Acromyrmex octospinosus and three colonies of A. echinatior. Control combinations, in which fungus and ants came from the same colony, run the diagonal of the ®gure and are accentuated with bold frames. Y-axes range from 0 mg to 110 mg in all ®gures and the fungus weight at day 0 is approximately 80 mg in all cases. Bars show the proportion (ranging from zero to one) of the 12 replicates in which the fungus had died completely by day 4 of the experiment. with all A. echinatior colonies. Barring the single and prob- models. Residual plots showed no indication of variance het- ably accidental death in the 33-48 combination, the only other eroscedasticity. Overall, the analyses of Table 1 show that cases where mortality of part of the fragments occurred where the major patterns visible in Figure 1 are highly signi®cant: combinations involving A. octospinosus colony 41, on one That material sampled from colonies of A. octospinosus is hand, and A. echinatior colonies 33, 47, and 48 on the other. equally likely to be compatible or incompatible with material The full model analyzing the data of Figure 1 explains from its own species or the other species A. echinatior, and 60% of the change in weight of the fungal fragments from A. echinatior shows a striking difference between interactions day 0 to day 4 (see Table 1). None of the species-level main with material from its own species and material from the factors are signi®cant, but both the colony-level effects and other species, A. octospinosus. the interactions are (P Ͻ 0.0001 in all cases). This indicates that interactions between ants from one colony and a fungal Genetic Similarity of Fungus Cultivars and fragment from another colony cannot be predicted from Ant-Fungus Incompatibility knowledge about the species of ant, without simultaneously taking the source of the fungus into account. However, grad- We compared the experimentally obtained fungus mortal- ually reducing the model to fewer factors showed that ant ity data with the genetic distances of the fungal clones in the species alone can explain almost 10% of the variation, which AFLP phylogeny of a much larger sample of Acromyrmex is the simplest possible signi®cant model (P Ͻ 0.0001), cultivars in the Gamboa area (see Fig. 2A). AFLP compar- whereas fungus ``species'' never has any signi®cant effect isons among the Acromyrmex fungus cultivars yielded a total (see Table 1). W-values of the Shapiro-Wilk tests are close of 56 bands that were polymorphic within the sample of to the expected value of one. This indicates that deviations isolates examined. Parsimony analysis of these data gave 896 from normality are small, although the large sample size equally most parsimonious trees of length 105 steps, CI ϭ makes these deviations signi®cant, particularly in the reduced 0.5243 (excluding uninformative characters), and RI ϭ 1986 A. N. M. BOT ET AL.

TABLE 1. Summary of the statistical analysis of the data of the transplant experiment illustrated in Figure 1. The ®rst column (source) gives the independent variables and their interaction terms. The following columns give the results (F-values with P-values in parentheses) of various general-linear-model analyses of the weight change of the fungal fragments between day 0 and 4. The Shapiro-Wilk test statistic for normality of residuals (W, with P-value in parentheses), the ®t of the entire model (F-values with P-values in parentheses), and the percentage of the total variance of the dependent variable explained by the model R2) are given at the bottom.

Plus ®rst-order Source Main effects only interaction Plus nested effects Full model

Fungus ``species'' 2.941,429 3.111,428 0.251,4 0.251,4 (0.0873) (0.0785) (0.6437) (0.6437)

Ant species 42.271,429 44.761,428 2.471,4 2.471,4 (Ͻ0.0001) (Ͻ0.0001) (0.1909) (0.1909)

Fungus ``species'' ϫ ant species 26.301,428 36.131,420 52.261,396 (Ͻ0.001) (Ͻ0.0001) (Ͻ0.0001)

Fungus colony within fungus ``species'' 17.134,420 24.784,396 (Ͻ0.0001) (Ͻ0.0001)

Ant colony within ant species 24.864,420 35.964,396 (Ͻ0.0001) (Ͻ0.0001)

Fungus colony within fungus ``species'' ϫ 8.8224,396 ant colony within ant species (Ͻ0.0001) W (Shapiro-Wilk test) 0.930 0.934 0.970 0.978 (Ͻ0.0001) (Ͻ0.0001) (Ͻ0.0001) (0.0267)

Fit of model (F) 22.602,429 24.723,429 24.5311,420 17.2035,396 (Ͻ0.0001) (Ͻ0.0001) (Ͻ0.0001) (Ͻ0.0001) R2 0.0953 0.1477 0.3912 0.6032

0.9030 (see Fig. 2A). The cultivars of the two sympatric these three colonies are almost identical, but 41 is an A. species of Acromyrmex leaf-cutting ants are represented on octospinosus colony, whereas 33 and 47 are A. echinatior all major branches of the cultivar phylogeny, and the two ant colonies. The other two clades in the phylogeny do not have species thus share all major fungal clones. The two plots that within-clade incompatibility and their colonies thus have val- resulted from the correspondence analysis (see Fig. 2B, C) ues close to zero on the horizontal axis (C2). are almost each other's mirror images, con®rming the ap- parent symmetry in the mortality results illustrated in Figure Proximate Factors Contributing to Incompatibility 1. The distances between colonies in the correspondence plots indicate how similar or different the observed incompatibility Incompatible ants and fungi became compatible after one patterns were. Compatible colonies with similar patterns of to two weeks of repeated and enforced exposure to the ini- incompatibility toward other colonies are close together in tially incompatible cultivar (see Fig. 3). During the ®rst days the correspondence plots. Colonies 48 and 31, for example, of exposure to a new cultivar, weight losses of the cultivar are the only colonies that are incompatible to colony 38 but fragments amounted to 65±100%, which in the previous ex- at least partially compatible to all other colonies. In the ant- periment implied complete incompatibility and rapid death fungus incompatibility plot (see Fig. 2B), these two colonies of the transplanted fungal fragment. Gradually, however, in- are identical in value, whereas there is a minor difference compatibility effects became less severe, and after approxi- between them in the fungus-ant incompatibility plot (see Fig. mately 10 days the daily weight losses of the introduced 2C), due to a single case of possibly accidental mortality in fungal fragments were no longer signi®cantly different from combinations between the fungus from colony 48 with the the controls. During the experiment the ants were regularly ants from the colonies 33 and 41 (see Fig. 1). observed to dissemble incompatible fungus in little pieces. The two axes presented in the plots of Figures 2B and 2C They would turn these pieces around between their front legs explain 95% of the variance in the data. The ®rst axis (C1) and chew on them with their mandibles, possibly actively describes the variation between groups, which corresponds killing the incompatible fungus. This behavior was never to differences between the major clades in the fungal phy- observed in the controls. logeny of Figure 2A. Colony 38, which is incompatible with Experimental application of fecal droplets to mycelia of all other colonies, has an opposite sign compared to the other nonresident fungi did not cause death of the arti®cial culture. ®ve colonies along the C1 axis. The fungi of colony 31 and However, cultures did appear to behave differently toward 48 are most distantly related to the fungus of colony 38, and fecal droplets that originated from resident host ants and they are most distant from this colony in the correspondence those that originated from the nonresident host ants (see Table plots. Colonies 33, 41, and 47 belong to an intermediate 2). Fecal droplets could either be absorbed or rejected by the branch in the phylogeny and also have an intermediate po- fungus. The fungus from colony 48 rejected most (75%) of sition in the correspondence plots. The second axis in Figures the droplets from ants of colony 38 and absorbed most (88%) 2B and 2C describes the variation within clades of the fungal of the droplets of its host workers. This matches the 100% phylogeny and is only variable for the clade containing col- mortality observed for this (cultivar 48/ants 38) combination ony 33, 41, and 47. Colony 41 comes out with a sign opposite in the transplant experiment (see Fig. 1). However, the fungus to colonies 33 and 47, which corresponds to the partial in- of colony 38 rejected and accepted as many droplets from compatibility between these colonies. The fungal clones of its own ants (50%) as from ants from colony 48 (50%). This INCOMPATIBILITY BETWEEN ANTS AND FUNGI 1987

FIG. 2. The phylogeny of the fungus cultivars of 16 Acromyrmex echinatior and 24 A. octospinosus colonies collected in Gamboa, Panama, in 1993, 1994, and 1996. (A) The tree is a midpoint rooted phylogram. The branch lengths are proportional to the number of changes in the AFLP markers, and the asterisks indicate bootstrap support greater than 50% (*), greater than 70% (**), or greater than 90% (***). Shaded boxes around colony numbers indicate the six colonies that were included in the exchange experiment and in the correspondence analysis. (B) The results of the correspondence analysis for the reactions of ants to fungi. (C) The same results for the reactions of fungi to ants. suggests that incompatibility is less pronounced in the re- plies, however, that the results of Table 2 would have been ciprocal (cultivar 38/ants 48) combination, which corre- signi®cant if the categories had been scored as ``normal ab- sponds again with the results given in Figure 1 (75% of the sorption'' and ``abnormal absorption.'' 12 transplanted fungal fragments died, but 25% maintained their original weight). The overall proportion of absorbed DISCUSSION droplets (see Table 2) for within-colony interactions was 69%, whereas the corresponding ®gure for across-colony in- The results of our study show that there is substantial ge- teractions was 37.5%. When tested by a logistic analysis netic variation among fungal cultivars reared by two sym- (JMP) this overall difference was marginally signi®cant patric species of leaf-cutting ants and that these two species (maximum-likelihood ␹2 ϭ 3.7; P ϭ 0.054). The fungus share all major clades of cultivars, indicating that horizontal quickly absorbed compatible droplets, but actively contained transfer does occasionally take place. Given the life-history incompatible droplets. This was done by growing hyphal tips characteristics of these basidiomycete symbionts, this vari- around the droplet, perpendicular to the surface, possibly to ation implies that host-symbiont con¯icts over symbiont mix- minimize contact at the interface. Contained droplets dried ing are unavoidable. We have extended a verbal model by up after a few days without being absorbed. Occasional ab- Frank (1996a) to derive predictions as to how these con¯icts sorption of droplets from alien ants would sometimes lead might be expressed and have con®rmed these predictions in to a dark red coloration of the PDA medium underneath the a series of experiments. Our ®ndings show that certain com- fungus. Such pigment accumulations often accompany in- binations of ants and cultivars are imcompatible. Genetic compatibility reactions of fungi both in vitro and in vivo, differences between cultivars can explain part of these in- but the effect could not be quanti®ed. This observation im- compatibily reactions, but incompatibility is mostly due to 1988 A. N. M. BOT ET AL.

FIG. 3. The proportion of fungus weight that was lost in combinations of fungus and ants from two incompatible colonies (Acromyrmex echinatior colony 48 and A. octospinosus colony 38) versus the controls that contained ants and fungus from the same colony. The value 100% corresponds to 0.5 g of fungus. Error bars represent standard errors (N ϭ 3). hostile behavior of the ants toward introduced cultivars, al- colony (who have no interest in the fruiting bodies of their though this behavior is possibly triggered by compounds cultivar) and the recipient ants (whose fungus gardens may from the fungus that are transferred via the alimentary tract suffer from virulence expressed by their cultivar after infec- of the ants. We also found that this imcompatibility is tran- tion with a novel strain), the ants are under constant selection sient and disappears after about 10 days. to suppress these tendencies in their cultivars. Due to the The experimentally observed incompatibility reactions extreme asymmetry in manipulative power between the host suggest that the success of horizontal transmission is likely and cultivar (sensu Frank 1997), the leaf-cutting ants seem to be low under normal ®eld conditions. However, we note to have won the evolutionary con¯ict with their cultivars and that some transplants in Figure 1 were fairly successful, even seem to have obtained a high degree of control. However, though they crossed ant-species barriers and despite geno- full control has not been achieved, because the fungus cul- typic differences (e.g., colonies 41 and 48; 31 and 47). This tivar is an ectosymbiont whose occasional spores or trans- would suggest that some horizontally transferred mycelial ported mycelial fragments can survive at least short periods fragments could possibly survive in other fungus gardens of transfer from one host to the other. Given the continuing without being recognized as a potential source of virulence interest of fungus cultivars in normal, vertical transmission by either the ants or the resident fungus. Applying an equiv- by ant gynes, this conceptual scenario would predict that rare alent of the Hamilton and May (1977) model, it would thus events of horizontal transmission are most likely to occur seem that natural selection may maintain some horizontal when an ant colony or fungus garden for some reason is transmission even in the higher leaf-cutting ants. This is be- doomed. In other words the (small) bene®ts of horizontal cause kin selection has both integrative and disruptive as- dispersal by the fungus would no longer be opposed by a pects, a phenomenon coined the paradox of kin selection by cost when future reproductive options via vertical transmis- Frank's (1997): The higher the relatedness of symbionts with- sion are lost but there is still the necessary mycelial mass to in single hosts, the more they also compete with relatives so grow a fruiting body. This may happen for example when a that some horizontal transmission is favored even if the suc- rare and virulent disease kills most of the worker force in a cess rate is very low. Because such horizontal transmission matter of days, or when the mother queen becomes senescent is normally detrimental for both the tending ants in a resident or dies at a time when none of the diploid brood can be raised into reproductive gynes. Note that only gynes are vectors of vertical transmission of the cultivar, so that the presence of TABLE 2. Rejection or absorption of fecal droplets by fungus of in- male brood is irrelevant from the perspective of the fungus. compatible colonies grown on arti®cial medium. Eight droplets were applied and the number of visible (unabsorbed) or absorbed droplets were scored after 24 h. Cultivar Polymorphism

Visible Absorbed Our results indicate that resident fungus cultivars have a Fungus origin Ant-feces origin droplets droplets signi®cant direct and indirect (via hostile ant behavior) com- Colony 38 colony 38 4 4 petitive advantage over nonrelated intruder cultivars. This Colony 38 colony 48 4 4 implies that, although potential competition between strains Colony 48 colony 38 6 2 of cultivars is common, actual cases of competition are prob- Colony 48 colony 48 1 7 ably rare. However, horizontal transmission is apparently still INCOMPATIBILITY BETWEEN ANTS AND FUNGI 1989 common enough for related sympatric species to share a com- with an increasing number of such loci on which they carry mon pool of cultivars (see Fig. 2A). This result matches different alleles (Hansen et al. 1993). Normally, mycelial earlier ®ndings for the lower attine ants, which share cultivar incompatibility patterns are correlated with relatedness be- lineages across distantly related species and genera (Mueller tween fungi (Worrall 1997), so that stronger reactions occur et al. 1998). It is less clear, however, what maintains the between more distantly related fungi. However, genomewide substantial populationwide genetic variation among fungus similarity estimated by AFLP markers is not necessarily an cultivars. Law's (1985) conceptual scenario of con¯ict-free accurate predictor of similarity at incompatibility loci. This mutualism in a homogeneous environment inducing a single may explain why the extent of incompatibility of the genet- symbiont genotype through positive frequency-dependent se- ically most distant colony (38) with colonies on the other lection is in fact the null hypothesis of the more modern major clades of the phylogeny of Figure 2A re¯ects the ge- pluralistic view of mutualistic interactions (e.g., Frank 1996a; nomewide genetic distances quite accurately, whereas the Bever 1999; Herre et al. 1999; Parker 1999; Thompson 1999). within-clade incompatibilities sometimes do not show such It is clear that the observed genetic diversity of cultivars with correlation. For example, the partial incompatibility pattern at least three major clades in our study population (see Fig. of colony 41 with colonies 47 and 33 is inconsistent with 2A) rejects that null hypothesis. However, our present data the absence of AFLP differences between the fungi involved, from just one population do not allow us to distinguish be- especially because colony 41 is mostly compatible with col- tween nonadaptive explanations invoking subdivided popu- onies 31 and 48, which belong to a different clade. An ex- lations and genetic drift versus adaptive explanations em- planation for this inconsistency could be that colony 33, 41, phasising negative frequency-dependent feedback mecha- and 47 were cultivated by different ant species (41 is A. nisms (Bever 1999) or geographic mosaics of locally co- octospinosus and 47 and 33 are A. echinatior). The slight adapted host-symbiont complexes (Thompson 1994, 1999; incompatibility of the ants of colony 41 with the fungus of Parker 1999). the third A. echinatior colony (48) corroborates the suggestion The extent of cultivar polymorphism obtained in our study that some degree of ant-species speci®city may exist, as is qualitatively matches the patterns of polymorphism found in also suggested by the overall statistical analysis (see Table other recent studies of mutualistic interactions (e.g., Wilkin- 1). However, the interaction between A. octospinosus colony son et al. 1996; Rowan 1998; Parker 1999). Although vari- 31 and A. echinatior colony 48, which have a genetically ation is substantial, speci®city is moderate at best, as the two identical cultivar (as do colonies 41 and 47), is unaffected Acromyrmex species share most if not all of the cultivar lin- by species speci®city. eages. However, it is important to note that the Acromyrmex Recognition and polymorphism are essential for genetic species do not share cultivar clones with sympatric or allo- coevolution of host-parasite interactions (Frank 1994), but patric Atta species (S. A. Rehner, U. G. Mueller, C. R. Currie, they seem equally important for mutualistic interactions as and T. M. Schultz, unpubl. ms.), so that speci®city at the long as these include aspects of mutual exploitation and vir- genus level seems to be upheld. Even within the genus Ac- ulence (Frank 1996a; this study). Horizontally transmitted romyrmex some speci®city may remain as the two species cultivars can only be eliminated by the host when they are are somewhat differently represented in the three major properly recognized. When symbionts are asexual and trans- clades of Figure 2A. The upper clade has a 3:1 ratio in favor mitted vertically, the interests in accurate recognition of host of A. octospinosus, the middle clade has a 1.3:1 ratio, and and resident symbiont are virtually completely aligned, so the lower clade a 1:1 ratio. Furthermore, colonies sampled that the resident symbiont is under selection to aid recog- in 1993 and 1994 (numbers 30 and below) make up 5/12 of nition, provided that the host has the power to cull unwanted the upper clade of Figure 2A, 3/14 of the middle clade, and symbionts. A possible mechanism serving this purpose in the 1/14 of the lower clade. These differences are not signi®cant, attine ant-fungus mutualism has been identi®ed in the present but do suggest that a larger-scale study of possible spatial study and warrants further investigation to understand its and temporal segregation of cultivars may be rewarding. details and to assess whether it also occurs in other symbioses with similar characteristics. Incompatibility in Free-living Basidiomycetes A recent experimental study on lower attines (Adams et and Cultivar Fungi al. 2001) has shown that Cyphomyrmex colonies whose fun- gus garden has been lost usurp neighboring colonies. The Free-living fungi usually defend themselves against fusion experiment was done with two sibling species that rear dis- with nonself mycelia by the mechanism of mycelial incom- tantly related fungi. Cultivar transfers between colonies could patibility (Leslie 1993; Anderson and Kohn 1995; Worrall be documented, corroborating inferences on occasional hor- 1997). Mycelial incompatibility is regulated by a multilocus, izontal transmission based on molecular data (Mueller et al. multiallele genetic system, where clones carrying identical 1998). However, the transfer success rate within ant species alleles at incompatibility loci are compatible and can fuse to was almost four times higher than between ant species, sug- form a single physiologically and genetically integrated my- gesting that incompatibility reactions might have occurred celium. In contrast, clones with different combinations of here as well. Our results also provide an interesting alter- alleles at these loci will be incompatible so that physiological native explanation to a result obtained by Rissing et al. fusion will not take place, either because the mycelia show (1989). In this study the desert leaf-cutting ant species Ac- an avoidance reaction or because of programmed cell death romyrmex versicolor was shown to have unrelated multiple of the hyphae in the contact zone. The intensity of incom- foundresses, where some foundresses seemingly voluntarily patibility between mycelia of basidiomycete fungi increases adopted more dangerous forager tasks. It would be interesting 1990 A. N. M. BOT ET AL. to investigate whether it was predominantly or exclusively LITERATURE CITED the foundress whose own fungus survived, who obtained the Adams, R. M. M., U. G. Mueller, A. K. Holloway, and A. M. Green. status of resident queen. Rissing et al. also observed that A. 2001. Garden sharing and garden stealing in fungus-growing versicolor workers had a tendency to raid brood, leaves, and ants. Naturwissenschaften 87:491±493. Anderson, J. B., and L. M. Kohn. 1995. Clonality in soilborne plant- fungus from each others colonies, but the frequencies of pathogenic fungi. Annu. Rev. Phytopathol. 33:369±391. transfers of these items across colonies was not reported. If Autuori, M. 1950. ContribuicËaÄo para o conhecimento da sauÂva(Atta raiding is indeed a universal phenomenon, the results of our spp., , Formicidae). V. NuÂmero de formas aladas study would make us expect that genetic variation among e rucËaÄo dos sauveiros iniciais. Arq. Inst. Biol. SaÄo Paulo 19: 325±331. fungal clones of A. versicolor may have become low due to Bekkevold, D., J. Frydenberg, and J. J. Boomsma. 1999. Multiple polygyny, so that incompatibility has become rare. However, mating and facultative polygyny in the Panamanian leafcutter if genetic variation among clones would prove to be com- ant Acromyrmex echinatior. Behav. Ecol. Sociobiol. 46:103±109. parable to what we found in Panamanian Acromyrmex, we Bever, J. D. 1999. Dynamics within mutualism and the maintenance of diversity: inference from a model of interguild frequency would expect that raiding of fungus fragments should be dependence. Ecol. Lett. 2:52±61. facultative and conditional on compatibility or should be fol- Bever, J. D., J. B. Morton, J. Antonovics, and P. A. Schultz. 1996. lowed by distinct intranidal nursing behavior of workers to Host-dependent sporulation and species diversity of arbuscular prevent mixing of cultivars. mycorrhizal fungi in a mown grassland. J. Ecol. 84:71±82. Bot, A. N. M., and J. J. Boomsma. 1996. Variable metapleural gland size-allometries in Acromyrmex leafcutter ants (Hymenoptera: Interactions with Other Symbionts Formicidae). J. Kans. Entomol. Soc. 69(suppl.):375±383. Boyd, N. D., and M. M. Martin. 1975. Faecal proteinases of the Interactions with other microorganisms associated with the fungus-growing ant, Atta texana: their fungal origin and eco- ant-fungus mutualism, such as the fungal parasite Escovopsis logical signi®cance. J. Insect Physiol. 21:1815±1820. (Currie et al. 1999b) and mutualistic Streptomyces bacteria Bronstein, J. L. 1994. Our current understanding of mutualism. Q. Rev. Biol. 69:31±51. (Currie et al. 1999a), may in¯uence the viability of fungus Chapela, I. H., S. A. Rehner, T. R. Schultz, and U. G. Mueller. fragments of different origin. The colonies that were used 1994. Evolutionary history of the symbiosis between fungus- for the present study had been in the laboratory for two years growing ants and their fungi. Science 266:1691±1695. prior to the experiments. They were thriving and continuously Cosmides, L. M., and J. Tooby. 1981. Cytoplasmatic inheritance and intragenomic con¯ict. J. Theor. Biol. 89:83±129. accumulating new fungus garden mass, characteristics that Currie, C. R. 2001. Prevalence and impact of a virulent parasite on are generally incompatible with Escovopsis infections (Currie a tripartite mutualism. Oecologia 128:90±106. 2001). Leaf-cutting ant colonies are apparently able to sup- Currie, C. R., and A. E. Stewart. 2001. Weeding and grooming of press this parasite under optimal laboratory conditions and pathogens in agriculture by ants. Proc R. Soc. Lond. B 268: 1033±1040. to remove it completely from their gardens after some time Currie, C. R., J. A. Scott, R. C. Summerbell, and D. Malloch. 1999a. when reinfections do not take place (Currie et al. 1999b). Fungus-growing ants use antibiotic-producing bacteria to control The Streptomyces bacteria, however, are always present on garden parasites. Nature 398:701±704. the cuticle of Acromyrmex workers. Their direct effect on the Currie, C. R., U. G. Mueller, and D. Malloch. 1999b. The agri- cultural pathology of ant fungus gardens. Proc. Natl. Acad. Sci. fungus cultivar is not yet known, but at present it seems USA 96:7998±8002. unlikely that these bacteria, which defend the fungus cultivar DoÈrfelt, H., and F. Creutzburg. 1994. Vom Pilz der Blattschneider- against Escovopsis infections (Currie et al. 1999a; C. R. Cur- Ameisen, Leucocoprinus gongylophorus. Boletus 18:29±35. rie, A. N. M. Bot, and J. J. Boomsma, unpubl. ms.), will Douglas, A. E. 1994. Symbiotic interactions. Oxford Univ. Press, Oxford, U.K. have a major effect on compatibility between strains of the Eberhard, W. G. 1980. Evolutionary consequences of intracellular fungus cultivar. However, surprises are possible, especially organelle competition. Q. Rev. Biol. 55:231±249. if it could be proven that the Streptomyces bacteria are also Ebert, D. 1994. Virulence and local adaptation of a horizontally occasionally horizontally transferred, have ample genetic transmitted parasite. Science 265:1084±1086. Ebert, D., and W. D. Hamilton. 1996. Sex againts virulence: the polymorphisms, and need to be controlled in ways that do coevolution of parasitic diseases. Trends Ecol. Evol. 11:79±82. not necessarily serve the interest of the fungus cultivar. Fisher, P. J., D. J. Stradling, and D. N. Pegler. 1994. Leucoagaricus basidiomata from a live nest of the leaf-cutting ant Atta ce- ACKNOWLEDGMENTS phalotes. Mycol. Res. 98:884±888. Frank, S. A. 1994. Recognition and polymorphism in host-parasite We thank D. Aanen, E. A. Herre, D. Molbo, U. G. Mueller, genetics. Phil. Trans. R. Soc. Lond. B. 346:283±293. D. R. Nash, and S. Rosendahl for fruitful discussion and ÐÐÐ. 1996a. Host symbiont con¯ict over the mixing of symbiotic lineages. Proc. R. Soc. Lond. B 263:339±344. comments on the manuscript; H. J. Carius for suggesting the ÐÐÐ. 1996b. Policing and group cohesion when resources vary. correspondence analysis; G. Nachman and H. C. Petersen for Anim. Behav. 52:1163±1169. advice on statistical analyses; M. Goldsmith and R. Gadeberg ÐÐÐ. 1997. Models of symbiosis. Am. Nat. 150:S80±S99. for help in maintaining laboratary colonies; the Smithsonian ÐÐÐ. 1998. Foundations of social evolution. Pp. 268 in J. R. Krebs and T. Clutton-Brock, eds. Monographs in behavior and Tropical Research Institute for providing logistic support and ecology. Princeton Univ. Press. Princeton, NJ. facilities to work in Gamboa; and the Autoridad Nacional del Hamilton, W. D. 1964. The genetical evolution of social behavior. Ambiente of the Republic of Panama for issuing collection I. J. Theor. Biol. 7:1±16. and export permits. The work of ANMB was funded by an ÐÐÐ. 1980. Sex versus non-sex versus parasite. Oikos 35: 282±290. EU±Marie Curie (B20) fellowship. Fieldwork was supported Hamilton, W. D., and R. M. May. 1977. Dispersal in stable habitats. by grants from the Carlsberg Foundation to JJB. Nature 269:578±581. INCOMPATIBILITY BETWEEN ANTS AND FUNGI 1991

Hansen, E. M., J. Stenlid, and M. Johansson. 1993. Genetic control Mueller, U. G., S. A. Rehner, and T. R. Schultz. 1998. The evolution of somatic incompatibility in the root-rotting basidiomycete Het- of agriculture in ants. Science 281:2034±2038. erobasidion annosum. Mycol. Res. 97:1229±1233. Mueller, U. G., T. R. Schultz, C. R. Currie, R. M. M. Adams, and Herre, E. A. 1999. Laws governing species interactions? Encour- D. Malloch. 2001. The origin of the attine ant-fungus mutualism. agement and caution from ®gs and their associates. Pp. 209± Q. Rev. Biol. 76:169±197. 237 in L. Keller, ed. Levels of selection in evolution. Princeton Parker, M. A. 1999. Mutualism in metapopulations of legumes and Univ. Press. Princeton, NJ. rhizobia. Am. Nat. 153:48±60. Herre, E. A., and S. A. West. 1997. Con¯ict of interest in a mu- Rehner, S. A., and G. J. Samuels. 1994. Taxonomy and phylogeny tualism: documenting the elusive ®g wasp seed trade-off. Proc. of Gliocladium analyzed from nuclear large subunit ribosomal R. Soc. Lond. B 264:1501±1507. DNA sequences. Mycol. Res. 98:625±634. Herre, E. A., N. Knowlton, U. G. Mueller, and S. A. Rehner. 1999. Rissing, S. W., G. B. Pollock, M. R. Higgins, R. H. Hagen, and D. The evolution of mutualisms: exploring the paths between con- R. Smith. 1989. Foraging specialization without relatedness or ¯ict and cooperation. Trends Ecol. Evol. 14:49±53. dominance. Nature 338:420±422. Hoekstra, R. F. 1987. The evolution of sexes. Pp. 59±91 in S. C. Rowan, R. 1998. Diversity and ecology of zooanthellae on coral Stearns, ed. Evolution of sex and its consequences. Birkhauser, reefs. J. Phycol. 34:407±417. Basel, Switzerland. Rowan, R., and D. A. Powers. 1991. A molecular genetic classi- Hurst, L. D. 1994. Cytoplasmatic genetics under inbreeding and ®cation of zooanthellae and the evolution of -algal sym- outbreeding. Proc. R. Soc. Lond. B 258:287±298. bioses. Science 251:1348±1351. Johnson, J. 1999. Phylogenetic relationships within Lepiota sensu Sanders, I. R., and A. H. Fitter. 1992. Evidence for differential lata based on morphological and molecular data. Mycologia 9: responses between host-fungus combinations of vesicular-ar- 443±458. buscular mycorrhyzas from a grassland. Mycol. Res. 96: Kaltz, O., and J. A. Shykoff. 1998. Local adaptation in host-parasite 415±419. systems. Heredity 81:361±370. SAS Institute. 1994. SAS/STAT user's guide. Vols. 1 and 2. SAS Law, R. 1985. Evolution in a mutualistic environment. Pp. 145± Institute, Inc., Cary, NC. 170 in D. H. Boucher, ed. The biology of mutualism: ecology Schultz, T. R., D. Bekkevold, and J. J. Boomsma. 1998. Acromyrmex and evolution. Croom Helm, London. insinuator new species: an incipient social parasite of fungus- Law, R., and S. Koptur. 1986. On the evolution of non-speci®c growing ants. Insectes Soc. 45:457±471. mutualism. Biol. J. Linn. Soc. 27:251±267. Seger, J. 1988. Dynamics of some simple host-parasite models with Leigh, Jr., E. G. 1999. Levels of selection, potential con¯icts, and more than two genotypes in each species. Phil. Trans. R. Soc. their resolution: the role of the `common good'. Pp. 15±30 in Lond. B 319:541±555. L. Keller, ed. Levels of selection in evolution. Princeton Univ. ÐÐÐ. 1992. Evolution of exploiter-victim relationships. Pp. 3± Press. Princeton, NJ. 25 in M. J. Crawley, ed. Natural enemies. Blackwell, London. Leslie, J. F. 1993. Fungal vegetative compatibility. Annu. Rev. Swofford, D. 1998. PAUP: phylogenetic analysis using parsimony. Phytopathol. 31:127±150. Beta ver. 4.0b2. Sinauer, Sunderland, MA. Lively, C. M. 1989. Adaptation by a parasitic trematode to local Thompson, J. N. 1994. The coevolutionary process. Univ. of Chi- populations of its snail host. Evolution 43:1663±1671. cago Press, Chicago, IL. ÐÐÐ. 1999. Migration, virulence and the geographic mosaic of ÐÐÐ. 1999. Speci®c hypothesis on the geographic mosaic of adaptation by parasites. Am. Nat. 153:34±47. coevolution. Am. Nat. 153:1±14. Lively, C. M., and M. F. Dybdahl. 2000. Parasite adaptation to Trivers, R. L. 1971. The evolution of reciprocal altruism. Q. Rev. locally common host genotypes. Nature 405:679±681. Biol. 46:35±57. Martin, M. M. 1992. The evolution of insect-fungal associations: Vos, P., R. Hogers, M. Bleeker, M. Reijans, and T. van de Lee. from contact to stable symbiosis. Am. Zool. 32:593±605. 1995. AFLP: a new technique for DNA ®ngerpringing. Nucleic Martin, M. M., N. D. Boyd, M. J. Gieselmann, and R. G. Silver. Acid Res. 23:4407±4414. 1975. Activity of faecal ¯uid of a leaf-cutting ant toward plant Weber, N. A. 1966. Fungus-growing ants. Science 153:587±604. cell wall polysaccharides. J. Insect Physiol. 21:1887±1892. ÐÐÐ. 1972. Gardening ants: the attines. American Philosophical Mintzer, A. 1990. Foundress female weight and cooperative foun- Society, Philadelphia, PA. dation in Atta leaf-cutting ants. Pp. 180±183 in R. K. Vander Wilkinson, H. H., J. M. Spoerke, and M. A. Parker. 1996. Diver- Meer, K. Jaffe, and A. Cedeno, eds. Applied myrmecology: a gence in symbiotic compatibility in a legume-Bradyrhizobium world perspective. Westview Press, San Francisco, CA. mutualism. Evolution 50:1470±1477. Muchovej, J. J., T. M. Della Lucia, and R. M. C. Muchovej. 1991. Worrall, J. J. 1997. Somatic compatibility. Mycologia 89:24±36. Leucoagaricus weberi sp. nov. from a live nest of leaf-cutting ants. Mycol. Res. 95:1308±1311. Corresponding Editor: K. Ross