Table 1.
Ant-associated and free-living actinomycete isolates used for the cross-phylogeny and within-Acromyrmex bioassay experiments.
Figure 1.
Schematic diagram of the assessment of actinomycete antagonism.
The resident actinomycete was inoculated in the center of a Petri dish and left at room temperature for three weeks, after which a suspension of the intruder actinomycete was inoculated to the entire unoccupied Petri dish area (top). One week after the intruder strain was applied, the minimum diameter of the resident strain and the minimum zone of inhibition (zoi) imposed on the intruder strains were measured. The bottom section of the figure shows four examples of actinomycete-actinomycete reactions, with no antagonism displayed by the residing clone towards the intruding actinomycete (top left), intermediate levels of antagonism with slight to strong inhibition (top right and bottom left), and to complete inhibition of the intruder by the residing strain (bottom right). Pictures are framed with colors according to the size of the zoi: white = no inhibition, light grey = 0.01–0.29 cm, grey = 0.30–0.59 cm, darker grey = 0.60–0.89 cm, and darkest grey>0.90 cm.
Table 2.
Relative genetic distances between pairs of actinomycetes involved in the across-phylogeny bioassay
Table 3.
Relative genetic distances between pairs of actinomycetes involved in the within-Acromyrmex bioassay
Figure 2.
Antagonistic reactions across the phylogeny of Pseudonocardia associated with attine ants.
Average degree of antagonism from the resident towards the intruder actinomycete for the 361 combinations of actinomycetes, spanning the attine phylogeny and including five free-living actinomycetes. Ant-associated Pseudonocardia strains are labeled with the genus and species name of the ants they were isolated from, and all strains are numbered as in Table 1 to distinguish strains originating from the same ant species. Different shades of grey denote strength of inhibition (average size of zone of inhibition, zoi; n = 3): white = no inhibition, light grey = 0.01–0.29cm, grey = 0.30–0.59cm, darker grey = 0.60–0.89cm, and darkest grey = above 0.90cm. A) Shows the bioassay results organized according to the phylogenetic placement of the nineteen strains paired. The phylogeny is based on 1393bp of 16S and 1004bp of EF-Tu. Bootstrap support values after 1000 pseudoreplicates under MP (top, left), ML (top, right), and NJ (bottom) conditions are given for the branches separating the major clades in the phylogeny (see text for details). B) Shows the same bioassay result organized according to the ant origin of the actinomycetes: leaf-cutting ants, Trachymyrmex ants, the lower attines, paleoattines, or free-living.
Table 4.
Statistical results of the generalized linear models with underlying gamma distributions for the two Petri dish bioassays.
Figure 3.
Antagonistic reactions between Pseudonocardia strains associated with Acromyrmex leaf-cutting ants.
Average degree of antagonism from the resident towards the intruder actinomycetes for the total of the 144 combinations performed within and between actinomycete strains isolated from five species of Acromyrmex. Strains are labeled after the ant species they were isolated from, and strains from the same ant species are distinguished by their labeling (A through L). Different shades of grey denote strength of inhibition (average size of zone of inhibition, zoi; n = 3): white = no inhibition, light grey = 0.01–0.29cm, grey = 0.30–0.59cm, darker grey = 0.60–0.89cm, and darkest grey = above 0.90cm. A) Shows the bioassay results organized according to the phylogenetic placement of the twelve strains paired, based on 1394bp of 16S and 959bp of EF-Tu sequences; bootstrap support values after 1000 pseudoreplicates under MP (top, left), ML (top, right), and NJ (bottom) conditions are given for the branches separating the major clades. B) Shows the bioassay results organized according to the ant species origin of the actinomycetes, with horizontal lines separating reactions displayed by isolates from the five ant species.