Figure 1.
Schematic overview of the three components of our study: the cross-phylogeny bioassay experiments (left panel), the within-Acromyrmex bioassay experiments (middle panel), and the sub-colony evaluation of the role of in vitro antagonism on stability of novel in vivo sub-colony ant-fungus-bacterium combinations.
For the cross-phylogeny bioassay experiments, the number of ant colonies for which cultivars and Pseudonocardia strains were obtained is indicated next to a schematic tree showing four of the five distinct attine agricultural systems: Paleoattine agriculture (pink), Neoattine lower agriculture (brown), Neoattine higher agriculture (green and purple), and leaf-cutter agriculture (purple) (see [30] for details). For the within-Acromyrmex bioassay experiments, the number of colonies from each of the five Acromyrmex species is likewise indicated. The right panel (sub-colony experiment) indicates the approach of the experiment: novel ant-fungus-bacterium combinations were performed in which there was directional antagonism from one, both, or none of the mutualists. The role of antagonism on stability was assessed by evaluating fungus garden mass and abundance of Pseudonocardia on ant cuticles (see main text for details).
Table 1.
Fungus-growing ant colonies from which Pseudonocardia and cultivar isolates were obtained.
Figure 2.
Micrographs showing typical extremes in reactions observed between symbiont pairings.
(a)–(d) Cultivar (middle) inhibition of Pseudonocardia (edge) (a) No cultivar-induced inhibition of Pseudonocardia, (b) intermediary cultivar-induced inhibition of Pseudonocardia, (c) strong cultivar-induced inhibition of Pseudonocardia, (d) no cultivar-induced inhibition of Pseudonocardia, but strong discoloration indicating antagonistic chemical interactions. (e)–(h) Pseudonocardia (middle) inhibition of the cultivar (edge). (e) No Pseudonocardia-induced inhibition of the cultivar, (f) intermediary Pseudonocardia-induced inhibition of the cultivar, (g) strong Pseudonocardia induced inhibition of the cultivar, and (h) weak Pseudonocardia-induced inhibition of the cultivar, but strong discoloration indicating antagonistic chemical interactions.
Table 2.
Combinations used in the sub-colony experiment evaluating whether the presence/absence of antagonism impacts the success of ant fungiculture.
Table 3.
Results of three ANOVAs testing the effects of ant origin, fungus origin, time since start of the experiment, directional antagonism from cultivars and Pseudonocardia, respectively, as well as the interaction between ant colony-of-origin and fungus colony-of-origin by time, on fungus weight (left) and actinobacterium coverage of ants (right).
Table 4.
Results of ANOVAs on each of the seven individual combinational experiments listed in Table 2.
Figure 3.
Diversity of interactions in bioassays examining the presence and degree of cultivar inhibition of Pseudonocardia for symbionts isolated from across the phylogenetic diversity of the ant-fungus-bacterium association.
Each box represents the average zone of inhibition (ZOI; n = 3) of a given pairing and different shades of grey indicate the degree of inhibition: White: ZOI = 0 cm, Light grey: ZOI = 0.01−0.29 cm, Grey: ZOI = 0.30−0.59 cm, Darker grey: ZOI = 0.60−0.89 cm, and Darkest grey: ZOI>0.90 cm.
Figure 4.
Diversity of interactions in bioassays examining the presence and degree of cultivar inhibition of Pseudonocardia between symbionts isolated from five species within the genus Acromyrmex.
Each box represents the average zone of inhibition (ZOI; n = 3) of a given pairing and different shades of grey indicate the degree of inhibition: White: ZOI = 0 cm, Light grey: ZOI = 0.01−0.29 cm, Grey: ZOI = 0.30−0.59 cm, Darker grey: ZOI = 0.60−0.89 cm, and Darkest grey: ZOI>0.90 cm. Frames around boxes indicate pairings in which dark coloration suggesting antagonistic chemical interactions between symbionts were observed.
Figure 5.
Diversity of interactions in bioassays examining the presence and degree of Pseudonocardia inhibition of cultivars for symbionts isolated from across the phylogenetic diversity of the ant-fungus-bacterium association.
Each box represents the average zone of inhibition (ZOI; n = 3) of a given pairing and different shades of grey indicate the degree of inhibition: White: ZOI<0.50 cm, Light grey: ZOI = 0.51−1.00 cm, Grey: ZOI = 1.01−1.50 cm, Darker grey: ZOI = 1.51−2.00 cm, and Darkest grey: ZOI>2.01 cm.
Figure 6.
The results of two bioassays examining the presence and degree of Pseudonocardia inhibition of cultivars between symbionts isolated from five species within the genus Acromyrmex.
Each box represents the average zone of inhibition (ZOI; n = 3) of a given pairing and different shades of grey indicate the degree of inhibition. (a) Shows the bioassay results for when Pseudonocardia was inoculated two weeks prior to the inoculation of the cultivar: White: ZOI<0.50 cm, Light grey: ZOI = 0.51−1.00 cm, Grey: ZOI = 1.01−1.50 cm, Darker grey: ZOI = 1.51−2.00 cm, and Darkest grey: ZOI>2.01 cm. (b) Shows the same bioassay pairings but in these pairings, the cultivar was inoculated one week prior to Pseudonocardia: White: ZOI = 0 cm, Light grey: ZOI = 0.01−0.29 cm, Grey: ZOI = 0.30−0.59 cm, Darker grey: ZOI = 0.60−0.89 cm, and Darkest grey: ZOI>0.90 cm. Frames around boxes indicate pairings in which dark coloration was observed suggesting antagonistic chemical interactions between symbionts.
Figure 7.
The results of the sub-colony experiment evaluating the effect of Pseudonocardia antagonism on sub-colony fungus garden weight.
Means±SE of three sub-colonies are given. During the first 10 days of the experiment, sub-colonies were provided 150±1 mg of fresh fungus material daily in order to allow sub-colony workers to accustom to the new fungus garden material. From day 11–33, the fungus garden mass was weighed, but not replaced with new fungus material. The direction of antagonism observed in vitro is indicated next to the legend, either as None (antagonism from neither symbiont), Cultivar (antagonism from the cultivar towards Pseudonocardia), Bacterium (antagonism from Pseudonocardia towards the cultivar), or Both (antagonism from both symbionts).
Figure 8.
The results of the sub-colony experiment evaluating the effect of cultivar antagonisms on the abundance of Pseudonocardia on the cuticle of sub-colony workers.
Means±SE of three sub-colonies, each containing three major workers carrying Pseudonocardia, are given, and values correspond to a scale from 0–12 [65]. The direction of antagonism observed in vitro is indicated next to the legend (see Fig. 7).