The population structure of antibiotic-producing bacterial symbionts of Apterostigma dentigerum ants: impacts of coevolution and multipartite symbiosis

Abstract

Fungus-growing ants (Attini) are part of a complex symbiosis with Basidiomycetous fungi, which the ants cultivate for food, Ascomycetous fungal pathogens (Escovopsis), which parasitize cultivars, and Actinobacteria, which produce antibiotic compounds that suppress pathogen growth. Earlier studies that have characterized the association between attine ants and their bacterial symbionts have employed broad phylogenetic approaches, with conclusions ranging from a diffuse coevolved mutualism to no specificity being reported. However, the geographic mosaic theory of coevolution proposes that coevolved interactions likely occur at a level above local populations but within species. Moreover, the scale of population subdivision is likely to impact coevolutionary dynamics. Here, we describe the population structure of bacteria associated with the attine Apterostigma dentigerum across Central America using multilocus sequence typing (MLST) of six housekeeping genes. The majority (90%) of bacteria that were isolated grouped into a single clade within the genus Pseudonocardia. In contrast to studies that have suggested that Pseudonocardia dispersal is high and therefore unconstrained by ant associations, we found highly structured ([Formula: see text]) and dispersal-limited (i.e., significant isolation by distance; [Formula: see text], [Formula: see text]) populations over even a relatively small scale (e.g., within the Panama Canal Zone). Estimates of recombination versus mutation were uncharacteristically low compared with estimates for free-living Actinobacteria (e.g., [Formula: see text] in La Selva, Costa Rica), which suggests that recombination is constrained by association with ant hosts. Furthermore, Pseudonocardia population structure was correlated with that of Escovopsis species ([Formula: see text], [Formula: see text]), supporting the bacteria's role in disease suppression. Overall, the population dynamics of symbiotic Pseudonocardia are more consistent with a specialized mutualistic association than with recently proposed models of low specificity and frequent horizontal acquisition.

Figure 1

Collection locations (A, B; see table 1 for definitions) and phylogenetic construction (C) of symbiotic Pseudonocardia. The phylogeny was based on a six-locus concatenated alignment and used Bayesian methods. The scale bar corresponds to 0.005 substitutions per site. Maximum likelihood methods yielded trees with similar topologies. Bayesian posterior probabilities are reported below branches, and bootstrap support values from maximum likelihood are reported above branches. Highlighted isolates are recombinant.

Figure 2

Phylogenetic analysis (maximum likelihood) of partial sequences from each of six housekeeping genes. The scale bar corresponds to 0.01 substitutions per site, and values at nodes report percentage bootstrap support. Topologies were largely congruent with the exception of three isolates that showed incongruence across clade 1 and clade 2, which suggests that they are recombinant. Note that 29 isolates, none of which showed incongruence, were removed for visual purposes. LS8 shows incongruence within gyrB, and isolates NPLR5 and SBCI13 show incongruence in dnaA. These isolates were also identified as recombinant using the Recombination Detection Program 3.44 software package and GENECONV (see “Material and Methods”); however, the overall recombination to mutation rate was marginal (ρ/θ = 0.09).

Figure 3

Isolation by distance in symbiotic Pseudonocardia in the Panama Canal Zone. Correlation between genetic and geographic distance was assessed using a Mantel test.

Figure 4

Association between the population genetic structure of symbiotic, antibiotic-producing Pseudonocardia associated with Apterostigma dentigerum in Central America (La Selva Biological Station, Costa Rica [LS-CR]; Barro Colorado Island, Panama [BCI-PA]; Pipeline Road, Panama [PLR-PA]; and Gamboa Forest, Panama [GAM-PA]) and two fungal symbionts (Escovopsis-pathogen and cultivar-mutualist). Population structure was congruent between Pseudonocardia and the Escovopsis pathogens they inhibit (A). No significant association was detected between Pseudonocardia and the cultivars that serve as the ant food source (B). Significance was assessed using a Mantel test. Escovopsis and cultivar Φ_ST values were taken from Gerardo and Caldera (2007).