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1
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ELECTRONIC SUPPLEMENTAL MATERIAL
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Hermógenes Fernández-Marín
Estrada
3
, Jelle van Zweden
2,4
1, 2, 3 *
, David R. Nash
2
, Sarah Higginbotham
3
, Catalina
, Patrizia d’Ettorre 2,5
, William T. Wcislo
3
, Jacobus J.
Boomsma
2
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12
1 Centro de Biodiversidad y Descubrimiento de Drogas, Instituto de Investigaciones Científicas y
Servicios de Alta Tecnología, Edificio 219, Ciudad del Saber, Clayton, Panamá City, Panamá
13
2
Centre for Social Evolution, Department of Biology, University of Copenhagen
14 Universitetsparken 15, DK-2100 Copenhagen, Denmark.
15 3
Smithsonian Tropical Research Institute, Box 0843-03092, Balboa, Ancón, Republic of
16 Panamá, or Unit 0948 DPO AA 34002-0948.
17 4
Laboratory of Socioecology and Social Evolution, Zoological Institute, University of Leuven,
18 Naamsestraat 59, box 2466, 3000 Leuven, Belgium
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20
5 Laboratoire d’Ethologie Expérimentale et Comparée (LEEC), University of Paris 13, Sorbonne
Paris Cité, France
21 *Corresponding author: HFernandez@indicasat.org.pa
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1

23
24 Nest collection
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26
Colonies were collected from 2004-2007 and transported to the University of Copenhagen, where they were maintained in temperature (ca 25 ºC) and humidity (ca 70%) controlled
27 rearing rooms (following Hughes & Boomsma 2006), and were used in experiments in
28 2007-2008. A second group of A. cephalotes colonies were collected in 2009, and
29 maintained in a laboratory at the Smithsonian Tropical Research Institute’s Gamboa facility
30 in Central Panama (STRI) using standard methods (Weber 1972). Subcolonies were
31 established following methods in Fernández-Marín et al. (2009). The numbers of ants per
32 subcolony are given in the sections below. Behavioral observations were made with a
33 stereomicroscope to obtain the frequencies of the different behaviors during five minutes
34 prior to infection, and for one hour after infection. To obtain Escovopsis strains from other
35 attine ants, colonies of 16 species were collected and maintained in Gamboa. The
36 Escovopsis strains were isolated from these colonies to obtain pure cultures following
37 methods described in (Fernández-Marín et al 2009). The strain of
Beauveria bassiana was
38 isolated from beetles by Professor Jørgen Eilenberg (Univeristy of Copenhagen).
39 Metarhizium was isolated from soil around Atta nests (Hughes & Boomsma 2004), named
40 Metarhizium anisopliae var anisopliae , but later classified by Rehner et al. (2010) as
41 Metarhizium brunneum
42
43 Chemical analyses by gas chromatography coupled with mass spectrometry (GC-MS)
44 Secretions were collected from the metapleural gland by inserting a capillary (diameter ca.
45 0.5 mm) through the meatus of the bulla. The secretions were then placed in vials with 25
2

46 μl of HPLC-grade pentane (Sigma–Aldrich, Denmark). We injected 2 µl of each extract
47 into an Agilent Technologies 6890N gas chromatograph, equipped with a HP-5MS capillary
48 column (30 m × 250 μm ID × 0.25 μm film thickness), a split-splitless injector and a 5375
49 Agilent Mass Spectrometer with 70 eV electron impact ionization. The carrier gas was
50
51 helium at 1 ml min
-1
. After an initial hold of 1 min at 50 ºC, the temperature rose to 300 ºC at a rate of 10 ºC min -1 , and then to 320 ºC at 20 ºC min -1
, with a final hold of 5 min at 320
52 ºC. The primary compound in the secretions (PAA) was identified by comparison of its
53 mass spectrum and retention index with those from an authentic reference sample. The
54 absolute concentration of PAA in infrabucal pellets was assessed by extracting 30 pellets
55 per treatment in 25 µl HPLC-grade pentane, containing 0.001 µg µl -1
pentadecane as an
56 internal standard, for 5 min. Peak areas were quantified using Agilent ChemStation v. 4 and
57 manual integration, and the absolute concentration of PAA was estimated by comparison of
58 the peak area to that of the internal standard.
59
60 Growth rates of fungal pathogens
61 All Escovopsis strains were grown and maintained in pure culture from which we obtained a
62 circular sample (ca. 0.5 cm in diameter) of Escovopsis, which was placed in the center of a petri
63 dish to inoculate it. We used 3 replicates of the same strain for each medium. Twenty days after
64 inoculation, we recorded the growth (diameter) of Escovopsis in each dish, and converted these
65 diameters to categorical data. Using similar methods, we also experimentally tested two strains
66 of Trichoderma (a fungal competitor within the fungus garden), a strain of Beauveria bassiana
67 and a strain of Metarhizium brunneum .
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72
68 Supplemental Results
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70
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Supplementary Figure 1. Samples of the growth rates of Escovopsis morphotypes from Atta ( Atta
02), Acromyrmex (Acro.2), Apterstigma (Aptero) y Cyphomyrmex (Cypho) tested to PDA, PDA +
DMSO, 100, 200, 400, 500, and 800 µg PAA (in PDA + DMSO), and cyclohexamide (100 µg).
4

73 Supplementary Table 1. Hygienic behavior of Atta cephalotes during infections with
74 pathogenic fungi. Comparisons are based on one-way ANOVA, with colony included as a
75 random effect. Values followed by the same letter are not significantly different based on
76 Tukey post-hoc tests.
F
4,16
8.71
4.44
0.667
0.770
ANOVA
P value
<0.001
0.013
0.624
0.561
77
78
Behavior
Control
Grooming 3.48 ± 2.5 A
MG
0 ± 0 A
Planting
Weeding
0 ± 0 A
0 ± 0 A
Mean number of behaviors per treatment
B. bassiana
10.9 ± 4.25 B
M. brunneum
5.49 ± 3.10 A,B 9.86 ± 6.60 B
Escovopsis 1
9.79 ± 2.21 B 10.36 ± 3.48 B 9.53 ± 3.58 B
8.46 ± 3.5 B
Escovopsis 2
7.36 ± 5.18 B
0.048 ± 0.11 A 0.08 ± 0.18 A
0.07 ± 0.09 A 0.45 ± 0.82 A
0.06 ± 0.13 A
0.18 ± 0.17 A
0 ± 0 A
0.58 ± 1.22 A
5

79 Supplementary Table 2. The presence of Phenylacetic Acid (PAA) on the legs of media workers
80 of Atta cephalotes , fungus garden fragments, isolates of pure fungal cultivars, fungal
81 pathogens, and infrabuccal pellets produced by workers of different size classes. Workers were
82 sampled without infection (No infection) and after infection.
Forelegs
Forelegs
Forelegs
Midlegs
Midlegs
Midlegs
Hindlegs
Hindlegs
Hindlegs
Fungus garden
Fungus garden
Fungus garden
Isolated fungus
Isolated fungus
Isolated fungus
Colonies
16 BB
19 BB
21 BB
16 BB
19 BB
21 BB
16 BB
19 BB
21 BB
16 BB
19 BB
21 BB
16 BB
19 BB
21 BB
Metarhizium brunneum
Beauveria bassiana
Escovopsis sp.
(from Atta cephalotes )
-
-
-
0/10
0/10
0/10
Pellets from minim workers 10,19, 21, x BB 11/11
Pellets from minor workers 10,19, 21, x BB 11/11
Pellets from media workers 10,19, 21, x BB 11/11
Pellets from major workers 10,19, 21, x BB 11/11
0/10
0/7
2/10
1/10
2/10
0/9
0/9
0/9
After Infection
4/10
1/10
3/7
0/10
0/10
0/7
0/10
-
-
-
-
-
-
-
-
-
-
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
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85
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87
88
89
90
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93
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95
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97
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102 Supplementary Table 3 . The presence or absence of Phenylacetic Acid (PAA) from
103 infrabuccal pellets collected after infection with Metarhizium brunneum (this study), and
104 the frequency of metapleural gland grooming rates after infection in different species of
105 attine ants, expressed as the number of grooming events per worker per hour after infection
106 (from Armitage et al. 2012).
107
Ant species
Mycocepurus smithii
Apterostigma collare
Aptersotigma goniodes
Myrmicocrypta ednaella
Cyphomyrmex longiscapus
Trachymyrmex cornetzi
Trachymyrmex zeteki
Trachymyrmex sp. 10
Trachymyrmex sp. 3
Sericomyrmex amabilis
Sericomyrmex cf amabilis
Acromyrmex echinatior
Acromyrmex octospinosus
Atta cephalotes
Atta sexdens
Atta colombica
Phenylacetic acid absent (0/5) absent(0/5) absent(0/5) absent(0/5) absent(0/5) absent(0/5) absent(0/5) absent(0/10) absent (0/5) absent(0/10) absent (0/10) absent (0/5) absent (0/5) present (5/5) present (5/5) present (5/5)
MG use
0 ± 0
0 ± 0
0 ± 0
0 ± 0
0.04 ± 0.07
0 ± 0
0.65 ± 0.68
2.22 ± 0.67
0 ± 0
8.66 ± 1.62
2.39 ± 0.34
1.68 ± 0.7
1.12 ± 1.3
13.5 ± 6.4
15.7 ± 2.0
16.6 ± 3.28
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108 References
Armitage SAO, Fernández-Marin H, Wcislo WT, Boomsma JJ. 2012 An evaluation of the possible adaptive function of fungal brood covering by attine ants. Evolution 66 , 1966-
1975.
Bischoff J.A., Rehner SA, Humber RA. 2009. A multilocus phylogeny of the Metarhizium anisopliae lineage. Micologia 101 , 512-530.
Fernández-Marín H, Zimmerman JK, Nash DR, Boomsma JJ, Wcislo WT. 2009 Reduced biological control and enhanced chemical pest management in the evolution of fungusfarming in ants. Proc R Soc B 276 , 2263-2269.
Hughes WOH, Boomsma JJ. 2006 Does genetic diversity hinder parasite evolution in social insect colonies? J Evol Biol 19 , 132-143.
Hughes WOH, Eilenberg J, Thomsen L, Boomsma JJ. 2004 Diversity of entomopathogenic fungi near leaf-cutting ant nests in a Neotropical forest, with particular reference to Metarhizium anisopliae var. anisopliae . J Inv Path 85 , 46-53
Weber NA 1972. Gardening ants: the attines, American Philosophical Society, PA.
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