16S <i>Pseudonocardia</i> phylogenetic tree.

<p>A phylogeny showing bacterial sequences associated with individual leaf-cutting ant colonies over time and their free-living relatives (<i>italics</i>). The phylogenetic tree was generated by the maximum likelihood method based on the Tamura-Nei model and bootstrapped 1000 times. The scale bar represents the genetic distance between samples, reflecting the number of nucleotide changes per site. The tree was created from 16 S rDNA sequences of <i>Pseudonocardia</i> exosymbionts obtained from leaf-cutting ants at the time of colony collection (regular text) and up to 9 years later (bold text). Year(s) of isolation noted in parenthesis after the colony code. Identical sequences obtained at different times are represented by a single sequence with multiple dates. Clades of <i>Pseudonocardia</i> associated with leaf-cutting ants are labeled with roman numerals <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103269#pone.0103269-Cafaro1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103269#pone.0103269-Poulsen4" target="_blank">[19]</a> and depicted in grey boxes. Ant-isolates are labeled by colony number, followed by the year isolated and GenBank identification number in parenthesis. The percentage sequence identity with original isolates is given in brackets after all re-isolates. Isolations from the same colony are boxed in the same color to facilitate comparison. Note that bacteria isolated from all colonies remain in the same phylogenetic clade over time, with the exception of a different morphological type (*) isolated once from a colony with two other independent re-isolates identical to the original sequence isolated.</p

Interaction between Workers during a Short Time Window Is Required for Bacterial Symbiont Transmission in <i>Acromyrmex</i> Leaf-Cutting Ants

<div><p>Stable associations between partners over time are critical for the evolution of mutualism. Hosts employ a variety of mechanisms to maintain specificity with bacterial associates. <i>Acromyrmex</i> leaf-cutting ants farm a fungal cultivar as their primary nutrient source. These ants also carry a <i>Pseudonocardia</i> Actinobacteria exosymbiont on their bodies that produces antifungal compounds that help inhibit specialized parasites of the ants' fungal garden. Major workers emerge from their pupal cases (eclose) symbiont-free, but exhibit visible Actinobacterial coverage within 14 days post-eclosion. Using subcolony experiments, we investigate exosymbiont transmission within <i>Acromyrmex</i> colonies. We found successful transmission to newly eclosed major workers fostered by major workers with visible Actinobacteria in all cases (100% acquiring, n = 19). In contrast, newly eclosed major workers reared without exosymbiont-carrying major workers did not acquire visible Actinobacteria (0% acquiring, n = 73). We further show that the majority of ants exposed to major workers with exosymbionts within 2 hours of eclosion acquired bacteria (60.7% acquiring, n = 28), while normal acquisition did not occur when exposure occurred later than 2 hours post-eclosion (0% acquiring, n = 18). Our findings show that transmission of exosymbionts to newly eclosed major workers occurs through interactions with exosymbiont-covered workers within a narrow time window after eclosion. This mode of transmission likely helps ensure the defensive function within colonies, as well as specificity and partner fidelity in the ant-bacterium association.</p></div

Acquisition source.

<p>a) Subcolony set-up. Newly eclosing <i>Acromyrmex</i> workers were fostered in subcolonies containing the following components 1) <i>Atta cephalotes</i> ants, a genus lacking visible symbiont, 2) minor workers (also lacking visible symbiont), 3) major workers without visible symbiont, 4) dissected thorax from major workers (changed every 48 hours) and minor workers (lacking visible symbiont) and 5) major workers with visible symbiont. Only pupae fostered with living major workers carrying <i>Pseudonocardia</i> developed symbiont coverage. b) The results of this experiment showed that <i>Acromyrmex echinatior</i> and <i>Ac</i>. <i>octospinosus</i> successfully acquired exosymbiotic bacteria only when pupae were raised in the presence of major workers carrying exosymbiont. Sample sizes (n) represent the number of focal ants surviving more than five days after eclosion. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103269#pone.0103269.s001" target="_blank">Figure S1</a> for mortality data. Photo of <i>Atta cephalotes</i> used with permission, ©Alex Wild, Schematic ant drawings modified from Poulsen et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103269#pone.0103269-Poulsen3" target="_blank">[18]</a>.</p

Acquisition timing.

<p>a) All set-ups contained one focal pupa, 4 minor workers and a piece of fungus garden. Major workers with visible exosymbiont were added at varying intervals from 0–1 hours to 24–30 hours post-eclosion. Controls contained major workers added at the time of subcolony set-up. Ants acquired in a gradient from 100% of controls to 0% of ants exposed after more than 2 hours. Drawings modified from Poulsen et al. 2003 and photos by S.E.M. b) Critical window period for exosymbiont acquisition in <i>Acromyrmex octospinosus</i>. The percentage of ants acquiring visible exosymbiont after exposure to exosymbiont-carrying majors introduced at varying times after eclosion. All pupae from 3–4 hour subcolonies died (n = 10), not shown. Sample sizes (n) represent the number of focal ants surviving more than five days after eclosion. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103269#pone.0103269.s001" target="_blank">Figure S1</a> for mortality data. *An additional 11 subcolonies were set up in the lab due to high field mortality.</p