PROCEEDINGS OF ' Proc. R. Soc. B (2007) 274, 1971-1978 THE ROYAL doi:10.1098/rspb.2007.0431 SOCIETY Published online 5 June 2007 Low host-pathogen specificity in the leaf-cutting ant-microbe symbiosis Stephen J. Taerumlz'T, Matias J. Cafaro11,3,4'3'4, Ainslie E. F. Little.1,2,3 Ted R. Schultz5 and Cameron R. Currie 1,2,3,$ 1Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045, USA 2Smithsonian Tropical Research Institute, Apartado 2072, Balboa, Republic of Panama 3 Department of Bacteriology, University of Wisconsin-Madison at Madison, Madison, WI53706, USA Departamento de Biologia, Universidad de Puerto Rico, Mayagiiez, PR 00681, USA 5Smithsonian Institution, National Museum of Natural History, PO Box 37012, Washington, DC 20013, USA Host-parasite associations are shaped by coevolutionary dynamics. One example is the complex fungus- growing ant-microbe symbiosis, which includes ancient host-parasite coevolution. Fungus-growing ants and the fungi they cultivate for food have an antagonistic symbiosis with Escovopsis, a specialized microfungus that infects the ants' fungus gardens. The evolutionary histories of the ant, cultivar and Escovopsis are highly congruent at the deepest phylogenetic levels, with specific parasite lineages exclusively associating with corresponding groups of ants and cultivar. Here, we examine host-parasite specificity at finer phylogenetic levels, within the most derived clade of fungus-growing ants, the leaf-cutters (Atta spp. and Acromyrmex spp.). Our molecular phylogeny of Escovopsis isolates from the leaf-cutter ant-microbe symbiosis confirms specificity at the broad phylogenetic level, but reveals frequent host-switching events between species and genera of leaf-cutter ants. Escovopsis strains isolated from Acromyrmex and Atta gardens occur together in the same clades, and very closely related strains can even infect the gardens of both ant genera. Experimental evidence supports low host-parasite specificity, with phylogenetically diverse strains of Escovopsis being capable of overgrowing all leaf-cutter cultivars examined. Thus, our findings indicate that this host-pathogen association is shaped by the farming ants having to protect their cultivated fungus from phylogenetically diverse Escovopsis garden pathogens. Keywords: coevolution; leaf-cutting ants; host-parasite interactions; host switching 1. INTRODUCTION common (Hafner et al. 1994; Clayton & Johnson 2003; Coevolutionary interactions shape the structure and diver- Gerardo et al. 2004). In addition, several parasites have sity of biological communities (Thompson 1994, 2005), wider host ranges than expected under coevolutionary and are theoretically predicted to lead to cospeciation theory (Harwood et al. 1998; Ricklefs et al. 2004). A further (Johnson et al. 2003). This expectation is especially pertinent expectation of host-parasite coevolution is that geographical for host-parasite interactions, where parasites depend on differentiation among parasites will reflect patterns of host their hosts for survival and reproduction, at the expense of differentiation across the geographical distribution, as host fitness (Price 1980). Hosts are selected to defend parasites are predicted to specialize on locally common against parasites, while parasites are selected to overcome host genotypes (Thompson 1994, 2005; Lively 1999; host defences (Dybdahl & Storfer 2003; Wegner et al. 2003). Dybahl & Storfer 2003; Lively et al. 2004). This coevolutionary arms race should result in congruent Host-parasite coevolutionary dynamics have been host and parasite phylogenies (Hafner & Nadler 1990; examined in the quadripartite attine ant-microbe symbiosis Page & Charleston 1998), where a parasite is highly (Currie et al. 2003; Gerardo et al. 2004). Attine ants (i.e. specialized for its matching host (Carius et al. 2001). These fungus-growing ants; Formicidae, Attini) are a monophy- expectations have been thoroughly examined in numerous letic group of 12 genera and over 210 described species host-parasite interactions (see reviews by Klassen 1992; (Holldobler & Wilson 1990; Schultz & Meier 1995), which Page 2003). have a geographical range from southern Argentina to Long Although there are many examples where there is some Island, USA (Weber 1972). Fungus-growing ants obligately degree of phylogenetic congruence between hosts and cultivate fungi for food (Weber 1972; Holldobler & Wilson parasites (Hafner & Nadler 1990; Schardl et al. 1997; 1990) by nourishing and fertilizing their fungal cultivars. Currie etal. 2003; Page 2003; Ricklefs etal. 2004; Weckstein The ant's fungal cultivars are frequently parasitized by 2004), perfect or near-perfect congruence is much less Escovopsis (Hypocreales: Ascomycota), a monophyletic genus of microfungi that directly consumes the cultivar (Currie et al. 1999a, 2003; Reynolds & Currie 2004). The * Author and address for correspondence: 420 Henry Mall, parasite is horizontally transmitted between nests (Currie Department of Bacteriology, University of Wisconsin at Madison, etal. 1999a), although the mechanisms by which this occurs Madison, WI 53706, USA. ([email protected]). ' Present address: Department of Biological Sciences, Dartmouth are unknown. Infections by Escovopsis result in a reduction in College, Hanover, NH 03755, USA. the growth rates of the fungus gardens, and occasionally Received 27 March 2007 1971 This journal is © 2007 The Royal Society Accepted 15 May 2007 1972 S. J. Taerum et al. Low host-parasite specificity ofEscovopsis force the ants to abandon their nests (Currie et al. 1999a; Province; and Rancho Frio in Darien Province), Argentina Currie 2001). To defend against Escovopsis, the ants employ (Parque Nacional Chaco and the road between Empedrado specific behaviours and have a mutualistic association with and Resistencia in Chaco Province; Parque Cruce Caballero, actinomycetous bacteria that produce antibiotics that Iguazii and San Pedro in Misiones Province), Ecuador (La specifically inhibit the specialized parasite (Currie et al. Selva Lodge and Biological Station and Tiputini Biodiversity 19996; Currie & Stuart 2001). Station in Orellana Province) and Guadeloupe (Basse-Terre The evolutionary histories of Escovopsis, cultivars and island) from 1997 to 2004. In most cases, ant nests were attine ants are highly congruent at the deepest phylogenetic excavated to sample Escovopsis directly from the fungus garden. levels (Currie et al. 2003). In contrast, Gerardo et al. (2004) In some cases, we isolated Escovopsis from colonies of demonstrated that Escovopsis strains from the nests of three A. colombica by collecting refuse material, following protocols sympatric species of Cyphomyrmex (a 'lower' genus of outlined by Currie (2001). In each case, up to 100 pieces of attines) are specifically associated with particular cultivar refuse or fungus garden from each nest were placed on potato strains, despite the incongruence in the evolutionary histories dextrose agar containing antibiotics (50mgl_1 each of of the ants and their fungal mutualists at lower (i.e. more penicillin and streptomycin). When mycelium of Escovopsis recent) phylogenetic scales for some 'lower' attines (Green appeared, it was sub-cultured onto fresh media and allowed to et al. 2002). However, since attine ants are very diverse, have grow (following the protocol of Currie et al. 1999a). In addition, a broad geographical distribution and vary in foraging and we obtained live cultures of Escovopsis weberi (an Escovopsis nest construction behaviours (Holldobler & Wilson 1990), it strain from an Atta sp. nest collected in Brazil) and Escovopsis is possible that different patterns of host-specificity occur aspergilloides (an Escovopsis strain from a Trachymyrmex ruthae across the phylogenetic diversity of attine ants. nest collected in Trinidad) from the Centraalbureau voor Here, we examine whether Escovopsis strains specialize Schimmelcultures (CBS 810.71 and 423.93). Samples of pure on particular species of leaf-cutting ants, a monophyletic culture Escovopsis were stored at — 20°C. DNA was extracted group within the 'higher' attines that only uses fresh plant following a CTAB protocol modified from Bender et al. (1983). material, mostly leaves, as substrate for cultivating their fungus garden (Schultz & Meier 1995; Wetterer et al. (b) Amplifications, sequencing and alignment 1998). The fungal mutualists cultivated by members of DNA sequencing targeted four exons and two introns of the the leaf-cutting ant genera Atta and Acromyrmex are nuclear elongation factor-1 a. (EF-la) gene, spanning a total of genetically and structurally distinct from the cultivars 1717 nucleotides. Three exons and both introns were farmed by other groups of attine ants (Chapela et al. amplified with primers EF1-3F (5'-CACGTCGACTCCGG 1994). In contrast to lower attines, which farm a wider CAAGTC-3') and EF1-5R1 (5-GTGATACCACGCTC diversity of cultivar strains (Mueller et al. 1998), leaf- ACGCTC-3'; Gerardo et al. 2004), and the fourth exon was cutting ants cultivate closely related fungal clones that are amplified with the Escovopsis-specific primers EF6-20F apparently derived from a single unique ancestor (Chapela (5'-AAGAACATGATCACTGGTACCT-3') and EF6-1000R et al. 1994), although recent research suggests that (5'-CGCATGTCRCGGACGGC-3'). Escovopsis DNA was horizontal transmission and recombination occur between diluted from its original concentrations to lower concentrations cultivars in different nests (Bot et al. 2001; Mikheyev