1
Mutualistic Fungi Control Crop Diversity in Fungus-Growing Ants
Supporting online material
1. Materials and Methods
In vitro culturing of fungus clones
Cultures of 42 fungal clones were obtained from colonies of two closely related (S1, S2)
sympatric species, A. echinatior and A. octospinosus, collected in Gamboa, Panama from
1994 to 2002. These ant species are phylogenetically separated, but cultivate the same
genetically diverse clade of fungi (Fig. S1). Colonies have since collection been kept in
climate rooms (60-70% RH; 25°C) at the Universities of Aarhus (1994 to 1999) and
Copenhagen (from 1999), on a diet of bramble leaves (Rubus fruticosus), rice, and fruit.
We inoculated 1-2 mm3 of mycelium from these fungus gardens onto Potato Dextrose
Agar (PDA, 39 g/l; Sigma-Aldrich, D-89552 Steinheim), and maintained these plates in
the dark at 25°C. Liquid cultures were subsequently made by transferring fungus
fragments (ca. 2 mm3) to sterile vials containing Potato Dextrose Broth (24 g PDB/l; ICN
Biomedicals, Inc., Ohio 44202, USA), which were likewise kept dark at 25°C.
Genetic comparison of fungus clones
DNA-extractions were obtained from the liquid cultures. To avoid any impact of the
medium, fungus material was spun down for 5 minutes at 6000 rpm and the supernatant
was removed. The DNeasy Plant Mini Kit (Qiagen GmbH, D-40724 Hilden) was used for
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DNA extraction. Amplified Fragment Length Polymorphism (AFLP) analysis, a standard
high resolution method for comparing total genome variation (S3), was performed as
described by Vos et al. (S4), using the protocol available at
http://wwwbio.leidenuniv.nl/~mollab/lab-users_info.html. Total DNA was cut with two
selective primer combinations (Eco-ACC X Mse-CAT and Eco-ACC X Mse-CAC) and
the amplification products were run on 5% polyacrylamide gels using an ABI-377
automatic sequencer, creating a gel image that was subsequently analysed using
GeneScan Analysis™ 3.1. All peaks above intensity 30 were scored, as this was the
threshold for the presence/absence of peaks being 100% repeatable in replicated runs of
the same sample (S5). A total of 85 bands (fragments of different length) were obtained.
Of these bands, 77 were polymorphic, so that they could be analysed as binary characters.
Scores were inserted into a data matrix to allow phylogenetic analysis and estimation of
the relative genetic distances between fungal clones. To do this, we used PAUP*4.0b10
(S6), which calculates the percentage of bands that are not homologous between pairs of
fungus clones (i.e. divides the number of band differences between pairs of clones by the
total number of bands).
Genetic comparison of multiple samples of fungus clones within colonies
Ten colonies (Ae33, Ae48, Ae109, Ae124, Ae132, Ao165, Ae168, Ao169, Ae191,
Ae210) were chosen to determine whether Acromyrmex ants rear single or multiple
fungus clones per colony. Five replicate isolations were made on separate agar plates
(using tufts obtained from different parts of the fungus gardens) and AFLP fingerprinting
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was done as described above. As a few of the isolates failed to amplify, we obtained a
total of 5-6 replicates per colony (mean = 5.7) after also including the initial AFLPsample that was used to characterize the clone for the first time. We subsequently
obtained sizes for all detectable bands in the AFLP profile by using GeneScan Analysis™
3.1, and we aligned the replicates per colony in Microsoft Excel, checking for any
mismatches, i.e. bands that were not detected by GeneScan Analysis™ 3.1 or were false
positives, on the original chromatograms. All AFLP patterns obtained from the same
fungus garden were 100% identical, whereas profiles from different fungus gardens
differed by at least 16 bands (24 on average).
To investigate the power of our conclusion that colonies rear only a single clone of
fungus, we calculated the probability of detecting multiple-clone colonies on the basis of
our obtained result that 5-6 replicates, within each of 10 colonies, were identical. This was
done on the basis of a binomial test and developed as a general model assuming that: i) If
there was more than one clone per colony, the number would be two, and ii) The
frequencies of the dominant and the alternative clone were constant across colonies. This
gives a detection probability (P) of:
N
P =1 - ∏ (f ni + (1-f)ni),
i=1
where N is the total number of colonies, n is the number of replicates in the ith colony,
and f and 1 – f denote the frequencies of the dominant and alternative clone within the
colony, respectively. Using an iterative procedure we calculated that double-clone
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colonies, with a frequency of the alternative clone as low as 4%, would have been
detected in our study with a power of 90%. Hence, we can safely assume that even if
colonies with multiple clones had escaped detection, the dominant clone would have a
frequency > 96%. This result rules out that the experimental colonies (a representative
sample of field colonies) had multiple fungus clones in reasonably balanced frequencies.
Additionally, all results of the present study and the previous study by Bot et al. (S7) rule
out that “second clones” can be maintained in fungus gardens at marginal frequencies.
In vitro incompatibility between plated fungus clones
In vitro fungus cultures from Acromyrmex echinatior (colony numbers 33, 48, 109, 145,
168, 197, 208, 209, 210) and A. octospinosus (colony numbers 35, 163, 169, 181, 198,
201, 205, GlP, AH5) were used to test whether plated fungi from different colonies were
(in)compatible. Fungus cultures from these colonies were paired in all possible (171)
combinations with 6 replicates for each combination (Fig. S2). This was done in Petri
dishes of 5 cm diameter containing PDA medium. Fungal tufts (1-2 mm3) were applied
1.5 cm from each other on the plates, which were kept in the dark at 25°C. Incompatibility
reactions were assessed two months after inoculation. Two months were needed because
these fungi grow only slowly on PDA medium and the mycelia had to have been in
contact for 1-2 weeks before we could unambiguously assess that observed interactions
were due to direct interaction between the mycelia. The semi-quantitative scale of
increasing rejection between mycelia was based on established practices (e.g. S8, S9): 0 =
demarcation zone absent, 1 = demarcation zone weak but present, 2 = demarcation zone
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broad and distinctly present, and 3 = strong demarcation zone with brown coloration of
mycelium and/or medium (cf. typically found mycelial incompatibility reactions in freeliving basidiomycetes: S10, S11) (Fig. 1B).
The association between relative genetic distance and mean incompatibility reaction
across replicates for any given combination was tested with a non-parametric Mantel test
of matrix correlation (S12) using the computer programme Arlequin 2.1 (S13). The
control pairings were not included to avoid biasing the test by a large number of
combinations with genetic distances and incompatibility scores of zero.
To test whether incompatibility reactions were affected by the ant species involved, we
first tested which of two possible ant species combinations was the correct one to use as a
main factor in the test. The first possibility was that a given fungus-fungus combination
was either one of fungi from the same ant species (Ae-Ae and Ao-Ao) or a fungus clone
from each of the ant species (Ae-Ao). Alternatively, the ant-species origin could be
divided in three groups, so that a given fungus-fungus combination was of fungi from Ae
only, Ao only, or a combination of the two (i.e. either Ae-Ae, Ae-Ao, or Ao-Ao). The
resolution of each of these two designs was tested with Contingency Analyses (S14). Both
showed an effect of species combination, having Likelihood Ratio χ2s of 416.9 (df = 3; P
< 0.0001) and 424.7 (df = 6; P < 0.0001) for two and three ant species combinations,
respectively. In order to test which of the two was the most accurate one to use, we tested
the χ2-distributions against each other using Microsoft Excel. This gave a marginal
significant difference (χ2 = 7.74; df = 3; P = 0.0516) in favour of using three groups (Ae-
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Ae, Ao-Ae, or Ao-Ao) in the final Effect Likelihood Ratio test (S15), where we
simultaneously tested the effect of genetic distance of fungal symbionts, and the
interaction effect of the two main factors on the fungus-fungus incompatibility reactions
observed. All tests were performed using the computer programme JMP (S16).
In vivo reactions of fungi and ants towards faecal droplets
To test the reactions of symbiont fungi towards faecal droplets of alien ants in vivo, we
used the A. echinatior colonies 33, 47, 144, 153, 168, 209 and the A. octospinosus
colonies 165 and 169 (Fig. S3). Faecal droplets were obtained from major workers (head
widths 2.0 - 2.9 mm) (S2), taken from the surface of fungus gardens, assuming that they
had recently fed on fungal material from their own garden. The ants were held with
forceps on a sterile glass plate and squeezed on the head and thorax until a droplet was
expelled. 0.5 µl autoclaved water was added to increase the volume of each droplet so that
it became easier to transfer in a sterile pipette.
For each of the original colonies, 64 small sub-colonies were set up, each containing 50 ±
2 mg (mean ± SE) of fresh fungus. These were placed in small pots (height 3.5 cm;
diameter 2 cm) with moist paper tissue at the bottom and a folded bramble leaf (the
normal leaf material used to maintain these colonies in the laboratory) (S7). Each subcolony was given 6 workers (4 minors and 2 majors) and was left for 24 hours for the ants
to acclimatize to the set-up. On the following day, slightly diluted faecal droplets (volume
1 µl; equivalent to 1.2 times the normal droplet volume) were applied directly to the
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fungus fragments of each sub-colony, so that each colony had 8 replicate control subcolonies with a droplet from their own ants and 8 sub-colonies challenged with droplets
from each of the seven foreign colonies. Twenty-four hours after droplet application (i.e.
within the time frame of normal absorption of compatible droplets; S17), fungus reactions
towards droplets were determined using a semi-quantitative scale of 6-categories: 0.
Complete absorption of the droplet; 1. Partial absorption of the droplet; 2. Distinct
mycelium growth on the droplet, but without absorption; 3. Absence of mycelial growth
in the surroundings of the droplet and no growth on the droplet surface; 4. Mycelial
avoidance of the droplet, with additional light to dark brown coloration of the droplet; and
5. Strong rejection, i.e. complete mycelial avoidance of the droplet and very dark
colouration of the droplet and the surrounding mycelium.
In the same experimental miniature colonies, the behavioural “weeding” response of the
ants towards the experimentally applied droplets was assessed by: 1. Scoring whether or
not the ants would actively remove droplets within 24 hours, and 2. Measuring the fresh
weight of the garbage material that the ants had accumulated at the bottom of their pot
during the 24 hours of the experiment. All possible associations between fungus
incompatibility reactions, droplet removal, mass of garbage deposited, and relative fungal
genetic distance were tested with Mantel tests of matrix correlation (S11). These tests
were performed on matrix values that were averages of all scores from reciprocal
combinations and of all replicates within these combinations (Fig. S2), as these were
never significantly different when tested with separate Mantel tests. As was done for the
fungus-fungus incompatibility reactions, we tested whether the results could be explained
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by the combination of ant species. Also here Likelihood Ratio tests and Contingency
Analyses (S13) showed that both designs with two and three ant-species treatment groups
(see above) were valid (χ2 = 112.1; df = 5; P < 0.0001 and 144.6; df = 10; P < 0.0001,
respectively) and once more the latter appeared to be superior (χ2 = 19.4; df = 5; P =
0.006). In the final Effect Likelihood Ratio test, we therefore simultaneously tested the
effect of ant species combination (Ae-Ae, Ao-Ae, or Ao-Ao), genetic distance between
resident and droplet-producing fungus, and the interaction between these two factors on
the droplet-incompatibility reactions observed (S14).
Transience of faecal droplet incompatibility
In an earlier study of the same two Acromyrmex species, it was shown that behavioural
hostility of ant workers towards alien fragments of fungus disappeared when the ants were
force-fed with the alien symbiont for 1-2 weeks (S6). For the present study, we tested
whether a similar period of force-feeding with a novel symbiont would also make faecal
droplets compatible, using two representative colonies, A. echinatior colony 209 and A.
octospinosus colony 165. Fungus clones from these two colonies were genetically highly
distinct (relative genetic distance = 0.36) and had, in the main experiment (Fig. 3),
displayed strong symmetrical fungus-droplet incompatibility (on average 4.6 ± 0.125
(SE)). Prior to the exchange experiment, droplets were obtained from six ants per colony,
three of which were tested on the resident fungus and three on the non-resident fungus,
using the same procedure and scale of incompatibility as described in previous sections.
Subsequently, four treatment groups were set up, each having six sub-colonies in which
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fungi and ants were paired, repeating the experimental setup that demonstrated the
transience in behavioural ant-fungus incompatibility performed by Bot et al. (S6). Each
sub-colony contained 50 mg of fungus, placed in small pots (height 3.5 cm; diameter 2
cm) with moist paper tissue at the bottom and a folded bramble leaf, two major workers,
and four minor workers (S6). During 10 days, a period chosen to match the mean number
of days of force-feeding required to eliminate behavioural incompatibility of ants and
foreign fungi (S6), sub-colony fungal mass was replaced daily by 50 mg of fresh fungus
from the source colony, so that also ants that had destroyed an incompatible fungus
fragment obtained new food. On day 10, faecal droplets were obtained from one major
worker, whose feeding had been manipulated for 10 days, and for one control worker per
sub-colony, and these droplets were tested for incompatibility on fungus from either
colony Ao165 or Ae209. These tests were done in new sub-colonies, each having 50 mg
of fungus and 4 minor workers taken directly from each of the original source colonies.
The incompatibility reactions were scored after 24 hours, i.e. 11 days after the initial
control sample, using the same scale as described above.
To statistically document the transience in faecal droplet incompatibility, we performed
an Effect Likelihood Ratio test (S14, S15) where degree of incompatibility was ordinal
and ant-fungus combination (Ao-Ao and Ae-Ae versus 2 x Ao-Ae), test-fungus (i.e. the
fungus clone that droplets were tested on, before and after force-feeding), and time
(before and after force-feeding) were nominal main factors. All statistical interaction
terms were included in the model, which showed that only the main effect of ant-funguscombination (χ2 = 9.498; df = 1; P = 0.0021) and the interaction between ant-fungus-
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combination and time (χ2 = 11.58; df = 1; P = 0.0007) were significant. The remaining
factors and interaction terms were not significant: test-fungus (χ2 = 0.0578; df = 1; P =
0.81), time (χ2 = 0.8204; df = 1; P = 0.3651), test-fungus x time (χ2 = 0.0578; df = 1; P =
0.81), ant-fungus combination x test-fungus (χ2 = 0.00052; df = 1; P = 0.9818), antfungus combination x test-fungus x time (χ2 = 0.00052; df = 1; P = 0.9818).
How fast is faecal-droplet incompatibility compared to mycelial incompatibility?
As it appeared that mycelial incompatibility reactions were considerably slower (often up
to two weeks after the mycelia had met when growing into each other on apar plates; Fig.
1) than faecal droplet incompatibility (<24 hours), we did an additional experiment to test
whether this difference was real or merely an artefact of the way in which we inoculated
our agar plates. We applied small tufts of fungus material directly on a mat of plated
fungus, testing 15 combinations that were known to be incompatible from the main
experiment. We monitored the plates daily and found that it took 3-7 days (mean ± SE:
5.6 ± 0.31) for incompatibility to be expressed. We thus conclude that incompatibility
reactions elicited by faecal-droplet are at least five times as fast as direct fungus-fungus
incompatibility reactions.
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2. Supplementary Figures
Figure S1
Expected phylogenetic relationships between Acromyrmex leaf-cutting ants and
their fungal cultivars under strict vertical transmission
Ant phylogeny
Fungus phylogeny
A.octospinosus
octospinosus
A.echinatior
outgroups
Phylogenetic relationships between the same ants and cultivars with
occasional horizontal transmission
Ant phylogeny
Fungus phylogeny
A.octospinosus
A.echinatior
outgroups
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Figure S2
Ae
33
Ae33
6
Ae
48
Ae48
6
6
Ae
109
Ae109
6
6
6
Ae
145
Ae145
6
6
6
6
Ae
168
Ae168
6
6
6
6
6
Ae
197
Ae197
6
6
6
6
6
6
Ae
208
Ae208
6
6
6
6
6
6
6
Ae
209
Ae209
6
6
6
6
6
6
6
6
Ae
210
Ae210
6
6
6
6
6
6
6
6
6
Ao
35
Ao35
6
6
6
6
6
6
6
6
6
6
Ao
163
Ao163
6
6
6
6
6
6
6
6
6
6
6
Ao
169
Ao169
6
6
6
6
6
6
6
6
6
6
6
6
Ao181
6
6
6
6
6
6
6
6
6
6
6
6
6
Ao
198
Ao198
6
6
6
6
6
6
6
6
6
6
6
6
6
6
Ao
201
Ao201
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
Ao
205
Ao205
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
Ao
GlP
AoGlP
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
Ao
AH5
AoAH5
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
Ao
181
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Colony number of droplet-receiving fungus
Figure S3
Colony number of droplet-producing fungus
Ae33 Ae47 Ae144 Ae153 Ae168 Ae209 Ao165 Ao169
Ae33
8
8
8
8
8
8
8
8
Ae47
8
8
8
8
8
8
8
8
Ae144
8
8
8
8
8
8
8
8
Ae153
8
8
8
8
8
8
8
8
Ae168
8
8
8
8
8
8
8
8
Ae209
8
8
8
8
8
8
8
8
Ao165
8
8
8
8
8
8
8
8
Ao169
8
8
8
8
8
8
8
8
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Supplementary Figure Legends
Figure S1. Schematic representation of the possible phylogenetic relationships between
sympatric Acromyrmex ant species and their fungal cultivars when transmission between
host generations is strictly vertical (top panel) and when occasional horizontal
transmission of symbionts between host generations occurs (bottom panel), drawn on the
basis of published phylogenies for the ants (S1, S2) and the fungi (S7; this study). The
dotted lines indicate which fungus clones are cultivated by each of the two ant species
with blue representing A. octospinosus and red A. echinatior. All empirical relationships
found in previous studies have shown that the bottom diagram, with partial incongruence
between the two phylogenies, is correct. This implies that we have two sympatric, nonhybridizing ant species, A. echinatior and A. octospinosus, that rear fungus clones of the
same genetically diverse clade, but without a clear subdivision into ant-species-specific
sub-clades. We hypothesize that horizontal transmission is relatively rare, because every
ant colony obtains its starting symbiont vertically and because the results obtained in the
present study show that switching symbionts is difficult. However, over evolutionary
time, such rare events can easily produce the kind of incongruent topologies shown at the
bottom.
Figure S2. The 18 x 18 experimental matrix showing the numbers of the colonies whose
fungi were combined in the mycelial incompatibility experiment (Fig. 1). Dark grey
shades indicate test pairings involving different colonies and light grey shades are control
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pairings. There were six replicates for each combination. Ae = Acromyrmex echinatior
and Ao = Acromyrmex octospinosus.
Figure S3. The 8 x 8 matrix showing the numbers of the colonies whose fungi were
combined in vivo with faecal droplets from other colonies. The two darker shades of grey
indicate reciprocal test pairings of fungi and droplets from different colonies, whereas the
light grey shades are control pairings. There were eight replicates for each combination,
but for the statistical analyses, the reciprocal combinations of fungus and droplets (the
two darker shades of grey) were combined (see also Materials and Methods). Ae =
Acromyrmex echinatior and Ao = Acromyrmex octospinosus.
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