Downloaded from rspb.royalsocietypublishing.org on 15 April 2009 
PROCEEDINGS THE ROYAL 
 OF  SOCIETY 
BIOLOGICAL 
SCIENCES 
No sex in fungus-farming ants or their crops 
Anna G. Himler, Eric J. Caldera, Boris C. Baer, Hermogenes Fernandez-Marin and Ulrich G. Mueller 
Proc. R. Soc. B published online 15 April 2009 
doi: 10.1098/rspb.2009.0313 
LL 
0 
Supplementary data 
References 
P<P 
Subject collections 
Email alerting service 
"Data Supplement" 
http://rspb.royalsocietypublishing.org/content/suppl/2009/04/08/rspb.2009.0313.DC1.ri 
tml 
This article cites 38 articles, 5 of which can be accessed free 
http://rspb.royalsocietypublishing.org/content/early/2009/04/08/rspb.2009.0313.full.ht 
ml#ref-list-1 
Published online 15 April 2009 in advance of the print journal. 
Articles on similar topics can be found in the following collections 
molecular biology (98 articles) 
developmental biology (57 articles) 
evolution (584 articles) 
Receive free email alerts when new articles cite this article - sign up in the box at the top 
right-hand corner of the article or click here 
LL 
0 
Advance online articles have been peer reviewed and accepted for publication but have not yet appeared in 
the paper journal (edited, typeset versions may be posted when available prior to final publication). Advance 
online articles are citable and establish publication priority; they are indexed by PubMed from initial publication. 
Citations to Advance online articles must include the digital object identifier (DOIs) and date of initial 
publication. 
To subscribe to Proc. R. Soc. 6 go to: http://rspb.royalsocietypublishing.org/subscriptions 
This journal is ? 2009 The Royal Society 
Downloaded from rspb.royalsocietypublishing.org on 15 April 2009 
PROCEEDINGS 
 OF ' 
THE ROYAL 
SOCIETY 
firstCite5 
e-publishing 
Proc. R. Soc. B 
doi:10.1098/rspb.2009.0313 
Published online 
0 
No sex in fungus-farming ants or their crops 
Anna G. Himler1'*, Eric J. Caldera1, Boris C. Baer2, 
Hermogenes Fernandez-Marin3'4 and Ulrich G. Mueller1'4 
1Section of Integrative Biology, Patterson Laboratories, University of Texas at Austin, Austin, TX 78712, USA 
School of Animal Biology and University of Western Australia, Crawley, Western Australia 6009, Australia 
3Department of Biology, University of Puerto Rico, San Juan, PR 00901, USA 
4
 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 fungus- 
growing 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. 
Keywords:  asexual; fungus-growing ants; symbiosis; Mycocepurus smithii; Wolbachia; thelytoky 
LL 
0 
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 evolutionary 
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-Marin  et al.   2005).   Moreover,  no 
* Author for correspondence (ahimler@email.arizona.edu). 
Electronic supplementary material is available at http://dx.doi.org/10. 
1098/rspb.2009.0313 or via http://rspb.royalsocietypublishing.org. 
males have been found in extensive nest excavations of 
M. smithii from throughout the Americas (Rabeling 
2004; Fernandez-Marin et al. 2005; Rabeling et al. 
2007), suggesting M. smithii may be parthenogenetic 
(Fernandez-Marin et al. 2005; see electronic supple- 
mentary 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 
March-April 2001, June 2002 and May 2003 in the Republic 
of Panama from five populations 50-150 km apart (Parque 
Soberania, Sherman Forest Reserve, or the Colon 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-Marin's survey of 228 male-less M. smithii nests 
in Puerto Rico (Fernandez-Marin et al. 2005). DNA samples 
were refrigerated in 95 per cent ethanol and extracted using 
Qiagen Dneasy kits. 
Received 23 February 2009 
Accepted 23 March 2009 This journal is ? 2009 The Royal Society 
Downloaded from rspb.royalsocietypublishing.org on 15 April 2009 
2    A. G. Himler et al.    Asexual fungus-farming ants 
LL 
0 
Figure 1. Reproductive tracts of Al. smithii and M. tardus queens, (a) The spermatheca of Al. smithii is translucent indicating that 
it is empty, (b) By contrast, a sperm-filled spermatheca the congener Af. tardus appears opaque, (c) Fully developed ovaries in 
both species contain mature eggs and several yellow bodies indicating the queens were active egg layers. The mussel organ, an 
internal lock structure of the female mating apparatus, is degenerate and unsclerotized in M. smithii compared to (d) the 
sclerotized functional mussel organ of Af. tardus. Scale bar, 0.25 mm. 
LL 
0 
(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 Cyphol5-16 (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 4-10 offspring per colony). DNA was extracted 
from single whole workers and queens' abdomens. Micro- 
satellite 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 15-16 
amplified ant DNA under the following PCR conditions: 
1 cycle of 94?C for 3 min; 35 cycles of 94?C for 40 s; 59?C for 
40 s; and 72?C for 30 s; and 1 final extension cycle at 72?C 
for 15 min. Each 10 ul PCR reaction contained: IX enzyme 
buffer (Promega); 3.75 mM MgCl2 (Promega); 0.25 mM of 
each dNTP (Promega); 0.25 U Taq DNA polymerase 
(Promega); 1 uM of each primer; and 1 ul DNA template at 
approximately 50 ng JJ.1     . 
(c) Female reproductive tract 
Colonies of Af. 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 Af. smithii for the presence of Wolbachia, Cardinium 
or other endosymbiotic bacteria by PCR (Holden et al. 1993; 
Zhou et al. 1998; Jeyaprakash & Hoy 2000; Zchori-Fein & 
Perlman 2004). DNA was extracted from individual workers 
or gyne abdomens for the survey with three Wolbachia-specific 
primers (Wenseleers & Billen 2000; Fournier et al. 2005; 
Pearcy et al. 2005), and from gyne larvae or adult ovaries for 
screens with Cardinium-specific and universal bacterial 
primers (Jeyaprakash & Hoy 2000; Zchori-Fein & Perlman 
2004). One to three workers, gynes or queens per colony were 
sampled from 15 colonies with a total of 31 samples; in only 
two colonies was a single ant tested. To verify PCR 
amplification, Formica truncorum ants and Drosophila simulans 
infected with Wolbachia served as positive controls and 
uninfected Formica sanguinae as the negative control. Twelve 
Af. smithii individuals were tested using primers Ch-F/Ch-R 
that amplify Cardinium and other related Bacteroidetes 
symbionts (Zchori-Fein & Perlman 2004), with the wasp 
Encarsia pergandiella as a positive control. Two universal 
bacterial primer pairs, U519F/U1406R and 338-356F/ 
16S-8R, were used to detect bacterial symbionts other than 
Wolbachia or Cardinium, with Escherichia coli as a positive 
control and sterile water as a negative control (Jeyaprakash & 
Hoy 2000; Baker et al. 2004). DNA quality of all samples was 
verified by successful amplification of the mitochondrial 
cytochrome oxidase I gene (Simon et al. 1994). 
(e) Antibiotic experiment 
Unmated virgin female M. smithii gynes were treated with 
antibiotics to test whether curing infection by potentially 
parthenogenizing microbes would permit male production. 
Thirty-five subcolonies were split from three source colonies. 
Subcolony replicates from the same source colony were 
blocked in groups of five for a total of seven groups. Each 
replicate subcolony contained a 20 mm3 garden fragment, 
Proc. R. Soc. B 
Downloaded from rspb.royalsocietypublishing.org on 15 April 2009 
Asexual fungus-farming ants    A. G. Himler et al.    3 
20 winged queens and approximately 75 workers, except one 
quintuplet set had six queens per subcolony. Within each 
group, replicates were randomly assigned to one out of four 
antibiotic treatments: 10 per cent streptomycin, 5 per cent 
penicillin, 5 per cent tetracycline, and 0.5 per cent rifampicin 
or a sucrose solution control. Particular antibiotic concen- 
trations were chosen because, in pilot experiments, females 
readily imbibed antibiotic sugar solutions at maximal 
concentrations without causing significant mortality (less 
than 10%) during a seven-day treatment, compared to control 
females kept without sugar solution that showed near 
100 per cent mortality. This difference in mortality demon- 
strated indirectly that the treated females were ingesting the 
antibiotic solutions. Replicate subcolonies were habituated 
for eight weeks in a two-chamber system before antibiotic 
treatment (Schultz 1993). During this time gardens doubled 
in size, and virgin females eclosed from pupae hidden in the 
transferred fungus garden, increasing the number of females 
per subcolony to an average of 36.5 ( + 7.6) that were all then 
treated with the assigned antibiotic. For treatment, all gynes 
were removed from each subcolony, and supplied with a drop 
of either 10 per cent (weight/volume) sucrose solution 
(control) or a 10 per cent sucrose solution laced with one of 
LL the four antibiotics. Fresh sucrose solutions were provided 
daily for seven days, and treated gynes were then returned to 
their subcolony. Subcolonies were maintained for 16 months 
until after the next round of annual gyne production was 
complete. Number of new queens produced after treatment 
was calculated per subcolony as number of live queens plus 
dead queens collected during the experiment, minus queens 
present at start of experiment. 
(f) Fungus switch experiment 
Seventy-five laboratory-reared, unmated gynes were randomly 
chosen from six M. smithii nests and placed individually on a 
brood-free fungus garden fragment (20 mm ) in a 6 cm Petri 
dish with a moistened plaster of Paris base. Thirty gynes each 
were randomly assigned to one of three garden types (w = 90 
queens total): native cultivar (from M. smithii), closely related 
cultivar (from Cyphomyrmex costatus), or distantly related 
cultivar (from Cyphomyrmex longsicapus). Replicates were 
maintained for 15 months over three generations and 
were fed weekly with a sterilized mix of polenta and ground 
oats as substrate for the fungus. Owing to non-normality, we 
examined the effect of fungal type on the number of workers 
and gynes produced using a Kruskal-Wallis test, and the effect 
of fungal type on queen survival using a Pearson's Chi-square 
test. All tests were conducted at the significance level of 0.05. 
3. RESULTS 
(a) Colony collections 
One hundred colonies of M. smithii collected from five 
populations in Panama produced over 10 000 new queens 
(gynes) during five years in the laboratory, an estimated 
10-20 times that number of workers, but no males. By 
contrast, approximately 30 other species of attine ants 
|_L maintained  in  the  same  laboratory  produced  males. 
Q Unmated M.   smithii queens  born  in  the  laboratory 
produced both worker and gyne offspring (but never 
males) when separated from their natal colony, starting 
clonal female lineages that could be propagated without 
mating over an indefinite number of generations. 
(b) Microsatellite fingerprinting and analysis 
Microsatellite DNA fingerprinting revealed that workers 
and gynes had an identical genotype to their mother in 12 
M. smithii colonies for which both queens and offspring 
were available. We genotyped nine heterozygous and three 
homozygous colonies. Under the assumption of sexual 
reproduction, the probability of heterozygous queens 
producing only heterozygous offspring in all nine colonies 
was (1/2)54 = 5.55X 10-17, therefore ruling out sexual 
reproduction (see electronic supplementary material, 
table 1). Another potential explanation for the observed 
genotype distributions (e.g. absence of homozygous 
offspring in nests with heterozygous queens) is strong 
selection against homozygotes at some pre-adult stage, but 
calculation shows that the strength of selection required is 
so extreme (selection coefficients greater than 0.94) that 
homozygotes should be very rare in the population 
(less than 2%). However, 25 per cent (3/12 colonies) of 
the genotyped colonies were homozygous. Therefore, the 
alternative explanation that homozygotes produced by a 
heterozygous queen are lethal can be ruled out. Offspring 
genotypes were always identical to their mothers' in both 
heterozygous and homozygous colonies, consistent with 
clonal reproduction. 
(c) Female reproductive tract 
Dissections of the female reproductive tracts of M. smithii 
queens collected from mature nests confirmed that they 
were never inseminated (empty spermathecae), although 
they had fully developed ovaries containing mature eggs 
and yellow bodies, indicating that they were active egg 
layers (figure 1). By contrast, M. tardus queens, a closely 
related species, had sperm-filled spermathecae. M. smithii 
queens also lack the 'mussel organ', a female reproductive 
structure found in other attine species into which the 
male's sclerotized genitalia lock during mating (figure 1; 
Baer & Boomsma 2006). 
(d) Molecular screens for endosymbiotic bacteria 
PCR screens for endosymbiotic Wolbachia, Cardinium and 
other bacteria in M. smithii were negative, ruling out 
infection with these bacteria as the cause of asexuality 
in M. smithii. 
(e) Antibiotic experiment 
As an additional test that bacteria might cause partheno- 
genesis in M. smithii, we treated 1320 gynes from 28 
experimental colonies with four different classes of 
antibiotics. Antibiotic purging of parthenogenizing 
bacterial symbionts reinstates male production in some 
asexual arthropods (Weeks et al. 2001; Stouthamer & Mak 
2002). However, the antibiotic-treated M. smithii queens 
produced 7488 daughter queens but no males during 
16 months post treatment (table 1). The combined 
molecular and antibiotic evidence therefore indicates 
absence of a male-eliminating bacterial endosymbiont 
in M. smithii. 
(f) Fungus switch experiment 
To test the hypothesis that asexuality is induced by the 
fungal cultivar we conducted a fungal-switch experiment 
in which M. smithii's normal fungus garden was replaced 
with a different fungus. Seventy-five newly emerged, 
unmated M.   smithii queens  were  isolated  either  on 
Proc. R. Soc. B 
Downloaded from rspb.royalsocietypublishing.org on 15 April 2009 
4    A. G. Himler et al.    Asexual fungus-farming ants 
Table 1. Antibiotic treatment of unmated queens. (Total number of queens produced (7844) over 16 months after antibiotic 
treatment. No males were produced. Concentrations were derived from similar experiments with wasps, and further tested in 
pilot experiments to maximize dose administered without causing significant mortality (up to 10%). Number of queens treated 
per subcolony represents the average + 1 s.d.) 
percentage of no. of queens 
antibiotic no. of treated per total no .of queens males 
antibiotic solution subcolonies subcolony queens treated produced produced 
streptomycin 10 7 37 + 17 264 931 0 
rifampicin 0.5 7 38 + 16 263 2560 0 
penicillin 5 7 34+16 236 1666 0 
tetracycline 5 7 36+12 249 1558 0 
control (sucrose) 10 7 47 + 16 332 1129 0 
total 1344 7844 0 
Table 2. Fungus switch experiment. (Seventy-five unmated M. smithii queens were placed either on their own fungus (control), 
a closely related or a distantly related fungus. No males were produced on any fungus type while female workers and new queens 
(gynes) were produced over three generations. There was no effect of fungus type on number of workers produced 
(Kruskal-Wallis test x!= 1.69, p = 0.4289), on the number of gynes produced (Kruskal-Wallis test x! = 0.0728, ? = 0.9643) or 
on queen survival (Pearson's Chi-square test %2 = 3.99, ? = 0.1363).) 
LL 
0 
offspring (F1+F2) 
fungus garden source 
M. smithii fungus 
(control fungus) 
C. costatus fungus 
(closely related 
fungus) 
C. longiscapus fungus 
(distantly related 
fungus) total 
no. of workers produced 77 
no. of gynes produced 7 
percentage queens reproducing 6/30 = 20% 
1 
3/30=10% 
128 
7 
6/30 = 20% 
213 
15 
15/90=17% 
LL 
0 
(i) their own fungus, (ii) a closely related fungus, or (iii) on 
a distantly related fungus. Surviving colonies were raised 
for two successive generations during which 21 unmated 
queens produced exclusively female offspring (213 Fl 
workers and 15 Fl gynes). These 15 gynes later produced 
17 F2 workers, but no males (table 2). Asexual 
reproduction by queens was independent of cultivar type 
(table 2), and cultivar substitution did not induce male 
production (electronic supplementary material). Most 
significantly, queens always produced workers before 
gynes, suggesting queen control over offspring caste, 
rather than extrinsic factors such as the cultivated fungus. 
4. DISCUSSION 
Six lines of evidence support complete and endogenous 
asexuality in M. smithii: absence of males, significant 
degeneration of the female mating apparatus, DNA 
fingerprint identity between mothers and daughters, 
absence of sex-ratio-biasing endosymbiotic bacteria, and 
the inability to induce male production by antibiotic 
treatment or fungal substitution. M. smithii represents, to 
our knowledge, the first case of a male-less species of ant, 
and the first case where females produce both reproduc- 
tive and worker offspring by asexual means. 
Degeneration of the female mating apparatus renders 
cryptic sex unlikely for M. smithii (Judson & Normark 
1996; Normark et al. 2003) and could suggest that the 
evolutionary origin of M. smithii from a sexual ancestor 
may not be very recent. A more remote rather than recent 
origin of asexuality is also consistent with the widespread 
Neotropical distribution of M. smithii from northern 
Argentina to northern Mexico. However, it is possible 
the degeneration of the unused mating apparatus could 
progress quickly. A coalescent analysis within a larger 
phylogenetic treatment of the Mycocepurus genus is 
needed to infer the age of asexuality in M. smithii. 
Asexuality in M. smithii could be caused by infection with 
endosymbionts such as Wblbachiabacteria (StouthamereraZ. 
1999), Cardinium bacteria (Zchori-Fein & Perlman 2004), 
undescribed microbes that can manipulate reproduction 
(Zchori-Fein & Perlman 2004), or by the fungal cultivar 
(Mueller 2002). Wolbachia has been found in several sexually 
reproducing ant species (Wenseleers et al. 1998) including 
fungus-growing ants (Van Borm et al. 2003), but is absent in 
seven partially asexual ant species (Wenseleers & Billen 
2000;FourniereJaZ. 2005; Pearcy etal. 2005). We document 
here that endosymbiotic microbes also appear to be absent in 
the asexual M. smithii. 
While asexual reproduction of haploid males developed 
from unfertilized eggs (arrhenotokous parthenogenesis) is 
a normal part of hymenopteran reproduction, asexual 
reproduction of diploid females from unfertilized eggs 
(thelytokous parthenogenesis) is exceedingly rare in ants. 
Only seven distantly related ant species produce females 
asexually (Wenseleers & Billen 2000; Fournier et al. 2005; 
Pearcy et al. 2005; Ohkawara et al. 2006) primarily by 
unmated workers, ranging from facultative asexual 
reproduction after queen death to nearly obligate asexu- 
ality in which the queen caste is absent or morphologically 
reduced (Itow et al. 1984; Schilder et al. 1999). However, 
males occur in all seven of these ant species, contrasting 
with the complete absence of males in M. smithii. Three 
distinct reproductive strategies of asexuality therefore 
appear to exist in ants: (i) worker reproduction of females 
with  a  trend  towards   queen  loss   (Messor capitatus, 
Proc. R. Soc. B 
Downloaded from rspb.royalsocietypublishing.org on 15 April 2009 
Asexual fungus-farming ants    A. G. Himler et al.    5 
0 
LL 
0 
Platytherea punctata, Cerapachys biroi, Pristomyrmex 
pungens; Pearcy et al. 2005), (ii) a mixed strategy where 
mated queens produce workers sexually but new queens 
asexually (Cataglyphis cursor, Wasmannia auropunctata, 
Vollenhovia emeryi; Fournier et al. 2005; Pearcy et al. 
2005; Ohkawara et al. 2006), and (iii) strict queen 
thelytoky with sterile workers (M. smithii). Pristomyrmex 
pungens was proposed as an obligate asexual since it 
reproduces by worker thelytoky; however, males with 
functional genitalia and normal spermatogenesis occur, 
indicating the potential for sex (Itow et al. 1984; Tsuji 
1988). By contrast, M. smithii has evolved a unique 
strategy in which queens produce both workers and new 
queens asexually, where workers are completely sterile 
(electronic supplementary material). 
Theory predicts that long-term asexuality increases 
extinction potential because absence of recombination 
constrains a species' ability to evolve, by generating novel 
genotypes and purging deleterious alleles at lower rates, 
eventually rendering asexual lineages inferior to competing 
sexual lineages (Barton & Charlesworth 1998). This cost of 
asexuality may be exacerbated in M. smithii through its 
dependence on asexual cultivar propagation (Mueller et al. 
1998, 2005). While cultivars of M. smithii are not ancient 
asexuals (Mueller et al. 1998; Mueller 2002), they are 
cultivated clonally within a nest and between nests over 
many years, making them vulnerable to fungal pathogens 
evolving within the gardens (Currie et al. 2003). Depen- 
dence of an asexual host on an asexual symbiont could 
therefore present a double cost of asexuality. Alternatively, 
the asexuality of both ant and fungus may permit the ants to 
fully exploit the evolutionary advantages of asexuality for 
unknown reasons. In cyclic parthenogens such as Daphnia 
and aphids, asexual forms predominate during resource 
abundance and switch to sexual reproduction when 
resources become limiting (Williams 1975; Bell 1982; 
Lynch 1984). However, M. smithii showed no switch to 
sexuality across seasonal differences, environmental 
gradients, geographical range (Argentina to Mexico), and 
diverse laboratory conditions studied to date. 
Assuming a double asexuality handicap, it is unclear 
how M. smithii could sustain one of the most widespread 
distributions and greatest local abundances of all attine 
ant species (Rabeling 2004; Rabeling et al. 2007). Strict 
clonal reproduction could eliminate kin-selected queen- 
worker conflicts that are thought to plague sexual insect 
societies (Ratnieks et al. 2006), and M. smithii therefore 
provides a unique experimental system to test theories of 
parent-offspring conflict and reproductive policing. 
In addition, unlike all other fungus-growing ant species 
that typically specialize on a narrow clade of fungi, 
M. smithii is the only attine ant species known to cultivate 
a diversity of fungi between different nests. This unpar- 
alleled diversity of cultivars propagated within M. smithii 
arises because M. smithii frequently switches to novel, 
distantly related fungal crops (Mueller et al. 1998; 
A. G. Himler & U. G. Mueller 2004, unpublished data). 
Frequent switching between fungal crops (symbiont 
reassociation) may mitigate the double asexuality handi- 
cap because switching generates novel combinations of ant 
farmer and crop genomes. This potentially creates 
sufficient variation in synergistic ant-crop phenotypes to 
outpace coevolution by crop pathogens (Mueller 2002; 
Van Doninck et al. 2002) and cope with environmental 
fluctuations. One possibility is that M. smithii is a 
geographic parthenogen (Vandel 1928), i.e. it tends to 
reproduce asexually in more extreme altitudes, further 
north, or more extreme environments than their sexual 
relatives (Bell 1982; Lynch 1984). However, M. smithii 
has one of the most extensive distributions of fungus- 
growing ants (Argentina to Mexico), and it is always 
sympatric with other, sexual attine species (except for 
some Caribbean island populations). Another possibility 
is that M. smithii can colonize a diversity of habitats 
because it represents a 'general purpose genotype' (GPG) 
able to tolerate broad environmental conditions (Lynch 
1984), an explanation applied to the ancient asexual 
darwinulid ostracods (Van Doninck et al. 2002). If so, 
M. smithii would be the first case of a symbiosis GPG and 
support the suggestion that clonality leads towards greater 
ecological generalization rather than specialization. 
The widespread distribution and ecological abundance 
of M. smithii challenge traditional views proposing that 
sexuality enables fungus-growing ants to assume the 
coevolutionary arms races of their asexual cultivars (i.e. 
effectively converting crop-pathogen arms races into races 
between ant farmers and crop pathogens; Herre et al. 
1999). Asexuality of M. smithii precludes such a 
hypothesized arms race transfer, and the ecological 
success of the dual asexual symbiosis between clonal ant 
farmers and their clonal crops therefore defies current 
theoretical expectations, perhaps adding a novel form of 
asexual scandal if M. smithii is shown to be of more ancient 
than recent evolutionary origin (Judson & Normark 1996; 
Normark et al. 2003). We predict that M. smithii will 
emerge not only as a new empirical system to test theories 
of parent-offspring conflict and policing in eusocial insects 
(Ratnieks et al. 2006), but also as a model permitting 
controlled symbiont-switch experiments in order to 
understand the evolutionary persistence of asexual 
lineages within a network of coevolving sexual pathogens 
and asexual mutualists. 
This work was supported by the NSF to AGH (DEB- 
0206372) and UGM (DEB-9983879, DEB-0110073) and 
the Section of Integrative Biology at UT Austin to AGH. 
B.C.B. was supported by the Swiss National Science 
Foundation (PA00A-104991) and the Danish Natural 
Science Research Council. We thank the Smithsonian 
Tropical Research Institute and the Autoridad Nacional del 
Ambiente of the Republic of Panama for collecting permits; 
T Wenseleers, K. Stanger-Hall and S. Perlman for Wolbachia 
and Cardinium controls; and B. Brandao for loaning 
Mycocepurus specimens from the Museu de Zoologia, Brazil. 
The authors are grateful to R. Adams, C. Currie, N. Gerardo, 
R. Koshy and A. Little for research assistance; C. Rabeling for 
sharing unpublished collection information; M. Florance and 
D. Hall for assistance with statistical analyses; and P. Abbot, 
N. Gerardo, B. Klein, C. Rabeling and A. Woods for helpful 
comments on the manuscript. 
REFERENCES 
Baer, B. & Boomsma, J. J. 2006 Mating biology of the leaf- 
cutting ants Atta columbica and A. cephalotes. J. Morphol. 
267, 1165-1171. (doi: 10.1002/jmor. 10467) 
Baker, G. C, Smith, J. J. & Cowan, D. A. 2004 Review and 
re-analysis of domain-specific 16S primers. J. Microbiol 
Methods 55, 541-555. (doi:10.1016/j.mimet.2003.08.009) 
Barton, N. H. & Charlesworth, B. 1998 Why sex and 
recombination? Science 281, 1986-1990. (doi: 10.1126/ 
science.281.5385.1986) 
Proc. R. Soc. B 
Downloaded from rspb.royalsocietypublishing.org on 15 April 2009 
6    A. G. Himler et al.    Asexual fungus-farming ants 
Bell, G.  1982  The masterpiece of nature: the evolution and 
genetics of sexuality. London, UK: Croom-Helm. 
Butlin, R. 2002 The costs and benefits of sex: new insights 
from old asexual lineages. Nat. Rev. Genet. 3, 311-317. 
(doi:10.1038/nrg749) 
Currie, C. R, Wong, B., Stuart, A. E., Schultz, T. R, Rehner, 
S. A., Mueller, U. G, Sung, G.-H., Spatafora, J. W. & 
Straus, N. A. 2003 Ancient tripartite coevolution in the 
attine   ant-microbe   symbiosis.   Science   299,   386-388. 
(doi:10.1126/science. 1078155) 
Fernandez-Marin, H., Zimmerman, J. K., Wcislo, W. T. & 
Rehner, S. A. 2005 Colony foundation, nest architecture 
and demography of a basal fungus-growing ant, Mycoce- 
purus smithii (Hymenoptera, Formicidae). J. Nat. Hist. 39, 
1735-1744. (doi: 10.1080/00222930400027462) 
Fournier, D., Estoup, A., Orivel, J., Foucaud, J., Jourdan, H., 
Le Breton, J. & Keller, L. 2005 Clonal reproduction by 
males  and  females  in  the  little  fire  ant.  Nature 435, 
1230-1234. (doi: 10.1038/nature03705) 
Herre, E. A., Knowlton, N., Mueller, U. G. & Rehner, S. A. 
1999 The evolution of mutualisms: exploring the paths 
between conflict and cooperation. Trends Ecol. Evol. 14, 
49-53. (doi:10.1016/80169-5347(98)01529-8) 
Holden, R R, Brookfield, J. F Y. & Jones, R 1993 Cloning 
and characterization of an/rsZhomologue from a bacterial 
LL symbiont of Drosophila melanogaster. Mol. Gen. Genet. 240, 
213-220. (doi: 10.1007/BF00277059) 
Itow, T, Kobayashi, K, Kubota, M., Ogata, K, Imai, H. T 
& Crozier, R. H.  1984 The reproductive cycle of the 
queenless   ant  Pristomyrmex pungens.   Insectes  Soc.   31, 
87-102. (doi: 10.1007/BF02223694) 
Jeyaprakash, A. & Hoy, M. A. 2000 Long PCR improves 
Wolbachia DNA amplification:  wsp sequences found in 
76% of sixty-three arthropod species. Insect Mol. Biol. 9, 
393-405. (doi: 10.1046/j. 1365-2583.2000.00203.x) 
Judson,  O.  R  & Normark,  B.  B.   1996  Ancient  asexual 
scandals. Trends Ecol. Evol. 11,41-46. (doi: 10.1016/0169- 
5347(96)81040-8) 
Lynch, M. 1984 Destabilizing hybridization, general-purpose 
genotypes and geographic parthenogenesis. Q. Rev. Biol. 
59,257-290. (doi:10.1086/413902) 
Mackay, W. R, Maes, J. M., Fernandez, R R. & Luna, G. 
2004   The   ants   of North   and   Central  America:   the 
genus Mycocepurus (Hymenoptera: Formicidae). J. Insect 
Sci. 4, 1-7. 
Mueller, U. G 2002 Ant versus fungus versus mutualism: 
ant-cultivar conflict and the deconstruction of the attine 
ant-fungus symbiosis. Am. Nat. 160, S67-S98. (doi: 10. 
1086/342084) 
Mueller, U. G, Rehner, S. A. & Schultz, T R. 1998 The 
evolution of agriculture in ants. Science 281, 2034-2038. 
(doi: 10.1126/science.281.5385.2034) 
Mueller, U. G, Gerardo, N. M., Annen, D. K, Six, D. L. & 
Schultz, T R. 2005 The evolution of agriculture in insects. 
Annu. Rev. Ecol. Evol. Syst. 36, 563-595. (doi:10.1146/ 
annurev.ecolsys.36.102003.152626) 
Normark, B. B., Judson, O. R & Moran, N. A. 2003 Genomic 
signatures of ancient asexual lineages. Biol. J. Linn. Soc. 
79, 69-84. (doi:10.1046/j.1095-8312.2003.00182.x) 
Ohkawara,  K,  Nakayama,  M.,  Satoh, A.,  Trindl, A.  & 
Heinze, J. 2006 Clonal reproduction and genetic caste 
differences in a queen-polymorphic ant, Vollenhovia emeryi. 
Biol. Lett. 2, 359-363. (doi:10.1098/rsbl.2006.0491) 
Pearcy,   M.,   Aron,   S.,   Doums,   C.   &   Keller,   L.   2005 
LL Conditional use of sex and parthenogenesis for worker 
and queen production in ants. Science 306, 1780-1783. 
(doi: 10.1126/science. 1105453) 
Rabeling, C. 2004 Nester, Streueintrag und symbiontische 
Pilze amazonischer Ameisen der Gruppe urspruenglicher 
Attini. MS thesis. University of Tubingen, Germany. 
Rabeling, C, Verhaagh, M. & Engels, W. 2007 Comparative 
study of nest architecture and colony structure of 
the fungus-growing ant species Mycocepurus goeldii and 
M. smithii (Formicidae: Attini). J. Insect Sci. 7, 40. 
Ratnieks, F L. W, Foster, K. R. & Wenseleers, T 2006 Conflict 
resolution in insect societies. Annu. Rev. Entomol. 51, 
581-608. (doi:10.1146/annurev.ento.51.110104.151003) 
Schilder, K, Heinze, J., Gross, R. & Holldobler, B. 1999 
Microsatellites reveal the clonal structure of populations of 
the thelytokous ant Platytherea punctata (F. Smith) 
(Hymenoptera; Formicidae). Mol. Ecol. 8, 1497-1507. 
(doi: 10.1046/j.l365-294x. 1999.00727.x) 
Schultz, T. R. 1993 Stalking the wild attine. Notes Undergr. 
{Mus. Comp. Zool., Harvard University) 8, 7-10. 
Simon, G, Frati, F., Beckenback, A., Crespi, B., Liu, H. & 
Flook, P. 1994 Evolution, weighting, and phylogenetic 
utility of mitochondrial gene sequences and a compilation 
of conserved PCR primers. Ann. Entomol. Soc. Am. 87, 
651-305. 
Stouthamer, R. & Mak, F. 2002 Influence of antibiotics on 
the offspring production of the Wolbachia-infected parthe- 
nogenetic parasitoid Encarsiaformosa. J. Invert. Pathol. 80, 
41-45. (doi: 10.1016/S0022-2011 (02)00034-4) 
Stouthamer, R., Breeuwer, J. A. J. & Hurst, G D. D. 1999 
Wolbachia pipientis: microbial manipulator of arthropod 
reproduction. Annu. Rev. Microbiol. 53, 71-102. (doi: 10. 
1146/annurev.micro.53.1.71) 
Tsuji, K. 1988 Obligate parthenogenesis and reproductive 
division of labor in the Japanese queenless ant Pristomyr- 
mex pungens. Behav. Ecol. Sociobiol. 23, 247-255. (doi: 10. 
1007/BF00302947) 
Van Borm, S., Wenseleers, T, Billen, J. & Boomsma, J. J. 2003 
Cloning and sequencing of wsp encoding gene fragments 
reveals a diversity of co-infecting Wolbachia strains in 
Acromyrmex leafcutter ants. Mol. Phylogenet. Evol. 26, 
102-109. (doi:10.1016/S1055-7903(02)00298-l) 
Van Doninck, K, Schon, I., De Bruyn, L. & Martens, K. 
2002 A general purpose genotype in an ancient asexual. 
Oecologia 132, 205-212. (doi: 10.1007/s00442-002-0939-z) 
Vandel, A. 1928 La parthenogenese geographique: contri- 
bution a retude biologique et cytologique de la partheno- 
genese naturelle. Bull. Biol. Fr. Belg. 62, 164-281. 
Villesen, P., Gertsch, P. J. & Boomsma, J. J. 2002 Microsatellite 
primers for fungus-growing ants. Mol. Ecol. Notes 2, 
320-322. (doi:10.1046/j.l471-8286.2002.00229.x) 
Weeks, A. R., Marec, F. & Breeuwer, J. A. J. 2001 A mite 
species that consists entirely of haploid females. Science 
292, 2479-2482. (doi: 10.1126/science. 1060411) 
Wenseleers, T & Billen, J. 2000 No evidence for Wolbachia- 
induced parthenogenesis in the social Hymenoptera. 
J. Evol. Biol. 13, 277-280. (doi:10.1046/j.1420-9101. 
2000.00168.x) 
Wenseleers, T, Ito, F., Van Borm, S., Huybrechts, R., 
Volkaert, F. & Billen, J. 1998 Widespread occurrence of 
the microorganism Wolbachia in ants. Proc. R. Soc. Lond. B 
265, 1447-1452. (doi:10.1098/rspb.l998.0456) 
Werren, J. H. & Windsor, D. M. 2000 Wolbachia infection 
frequencies in insects: evidence of a global equilibrium? 
Proc. R. Soc. Lond. B267, 1277-1285. (doi:10.1098/rspb. 
2000.1139) 
Williams, G. C. 1975 Sex and evolution. Princeton, NJ: 
Princeton University Press. 
Zchori-Fein, E. & Perlman, S. J. 2004 Distribution of the 
bacterial symbiont Cardinium in arthropods. Mol. Ecol. 13, 
2009-2016. (doi:10.1111/j.l365-294X.2004.02203.x) 
Zhou, W, Rousset, F. & O'Neill, S. 1998 Phylogeny and 
PCR-based classification of Wolbachia strains using wsp 
gene sequences. Proc. R. Soc. Lond. B 265, 509-515. 
(doi: 10.1098/rspb. 1998.0324) 
Proc. R. Soc. B