No sex in fungus-farming ants or their crops

Anna G. Himler () 2 Eric J. Caldera 2 Boris C. Baer 1 Hermo genes Ferna ndez-Marn 0 3 Ulrich G. Mueller 2 3 0 Department of Biology, University of Puerto Rico , San Juan, PR 00901 , USA 1 School of Animal Biology and University of Western Australia , Crawley, Western Australia 6009 , Australia 2 Section of Integrative Biology , Patterson Laboratories , University of Texas at Austin , Austin, TX 78712 , USA 3 Smithsonian Tropical Research Institute , Apartado 2072, Balboa-Ancon , Republic of Panama Asexual reproduction imposes evolutionary handicaps on asexual species, rendering them prone to extinction, because asexual reproduction generates novel genotypes and purges deleterious mutations at lower rates than sexual reproduction. Here, we report the first case of complete asexuality in ants, the fungusgrowing ant Mycocepurus smithii, where queens reproduce asexually but workers are sterile, which is doubly enigmatic because the clonal colonies of M. smithii also depend on clonal fungi for food. Degenerate female mating anatomy, extensive field and laboratory surveys, and DNA fingerprinting implicate complete asexuality in this widespread ant species. Maternally inherited bacteria (e.g. Wolbachia, Cardinium) and the fungal cultivars can be ruled out as agents inducing asexuality. M. smithii societies of clonal females provide a unique system to test theories of parent-offspring conflict and reproductive policing in social insects. Asexuality of both ant farmer and fungal crop challenges traditional views proposing that sexual farmer ants outpace coevolving sexual crop pathogens, and thus compensate for vulnerabilities of their asexual crops. Either the double asexuality of both farmer and crop may permit the host to fully exploit advantages of asexuality for unknown reasons or frequent switching between crops (symbiont reassociation) generates novel ant-fungus combinations, which may compensate for any evolutionary handicaps of asexuality in M. smithii. 1. INTRODUCTION The vast majority of eukaryotes reproduce sexually. Multicellular asexuals are rare, occur sporadically across the tree of life, and, with a few notable exceptions ( Judson & Normark 1996; Butlin 2002), are thought to be short-lived descendents derived recently from sexual ancestors (Barton & Charlesworth 1998). Theory predicts asexuality is advantageous because asexual lineages should out-compete sexual ones by circumventing the costs of sex (e.g. cost of meiosis, mating effort and producing males), however asexuality is thought to be evolutionarily disadvantageous because it purges deleterious mutations and generates novel genotypes more slowly than sexual reproduction (Butlin 2002). However, the pervasiveness of sex among multicellular organisms suggests that the advantages outweigh the costs (Barton & Charlesworth 1998). The real evolutionary conundrum, therefore, is not the pervasiveness of sexual lineages, but the persistence of some asexual lineages over extended evolutionary time ( Judson & Normark 1996; Herre et al. 1999). Similar to all other fungus-growing ants in the strictly Neotropical tribe Attini, Mycocepurus smithii (Formicidae, Attini) obligately farms basidiomyete fungi for food (Mueller et al. 1998). M. smithii has one of the widest distributions of any fungus-growing ant, ranging from Mexico and the Caribbean to Argentina (Mackay et al. 2004; Fernandez-Marn et al. 2005). Moreover, no males have been found in extensive nest excavations of M. smithii from throughout the Americas (Rabeling 2004; Fernandez-Marn et al. 2005; Rabeling et al. 2007), suggesting M. smithii may be parthenogenetic (Fernandez-Marn et al. 2005; see electronic supplementary material). As in other Hymenoptera (Werren & Windsor 2000), asexuality in M. smithii could be caused by infection with endosymbionts such as Wolbachia bacteria (Stouthamer et al. 1999), or by the vertically transmitted exosymbiont (e.g. the fungal cultivar; Mueller 2002). Here, we test the hypothesis that M. smithii is asexual using genetic, morphological and experimental analyses. 2. MATERIAL AND METHODS (a) Colony collections All M. smithii colonies in this study were collected in MarchApril 2001, June 2002 and May 2003 in the Republic of Panama from five populations 50150 km apart (Parque Soberana, Sherman Forest Reserve, or the Col on Province), Colonies were maintained in the laboratory for up to four years and never produced males. Field surveys in Panama (100 nests; AGH & UGM), Guyana (5 nests; UGM), Ecuador (6 nests; AGH), Peru (20 nests; C. Rabeling 2004, personal communication), Argentina (7 nests; UGM), and Brazil (132 garden chambers from an unknown number of neighbouring nests; Rabeling 2004; Rabeling et al. 2007) failed to find any males in M. smithii, complementing Fernandez-Marns survey of 228 male-less M. smithii nests in Puerto Rico (Fernandez-Marn et al. 2005). DNA samples were refrigerated in 95 per cent ethanol and extracted using Qiagen Dneasy kits. (b) Genotyping: DNA extraction and microsatellite amplification To test whether M. smithii offspring were clones of their mothers, we screened 14 microsatellite primer pairs developed for other fungus-growing ant genera (Villesen et al. 2002). Thirteen of these loci either did not amplify or were monomorphic, and thus uninformative. Using the single informative locus Cypho1516 (two alleles; 150, 152 bp), we genotyped 66 M. smithii specimens, from 12 Panamanian colonies for which both queen and workers were available (queen and 410 offspring per colony). DNA was extracted from single whole workers and queens abdomens. Microsatellite PCR products were run on an ABI 3100 automated sequencer and analysed using GENESCAN v. 3.7 and GENOTYPER v. 3.6. Microsatellite primer Cypho 1516 amplified ant DNA under the following PCR conditions: 1 cycle of 948C for 3 min; 35 cycles of 948C for 40 s; 598C for 40 s; and 728C for 30 s; and 1 final extension cycle at 728C for 15 min. Each 10 ml PCR reaction contained: 1X enzyme buffer (Promega); 3.75 mM MgCl2 (Promega); 0.25 mM of each dNTP (Promega); 0.25 U Taq DNA polymerase (Promega); 1 mM of each primer; and 1 ml DNA template at approximately 50 ng mlK1. (c) Female reproductive tract Colonies of M. smithii and Mycocepurus tardus were excavated and six resident non-winged queens of each species were dissected to inspect their reproductive organs and determine reproductive status. Mated, reproductively active queens are characterized by: (i) presence of sperm in the spermatheca (empty spermatheca appears translucent grey; sperm-filled spermatheca appears opaque white; figure 1), (ii) fully developed ovaries containing mature eggs in the ovarioles, and (iii) the presence of yellow bodies in the ovarioles. Yellow bodies (remnants of follicular epithelium) indicate that the ant had laid eggs. (d) Molecular screens for endosymbiotic bacteria We tested M. smithii for the presence of Wolbachia, Cardinium or other endosymbiotic bacteria by PCR (H (...truncated)