Local Adaptation of Bacterial Symbionts within a Geographic Mosaic of Antibiotic Coevolution

Abstract

The geographic mosaic theory of coevolution (GMC) posits that coevolutionary dynamics go beyond local coevolution and are comprised of the following three components: geographic selection mosaics, coevolutionary hot spots, and trait remixing. It is unclear whether the GMC applies to bacteria, as horizontal gene transfer and cosmopolitan dispersal may violate theoretical assumptions. Here, we test key GMC predictions in an antibiotic-producing bacterial symbiont (genus Pseudonocardia) that protects the crops of neotropical fungus-farming ants (Apterostigma dentigerum) from a specialized pathogen (genus Escovopsis). We found that Pseudonocardia antibiotic inhibition of common Escovopsis pathogens was elevated in A. dentigerum colonies from Panama compared to those from Costa Rica. Furthermore, a Panama Canal Zone population of Pseudonocardia on Barro Colorado Island (BCI) was locally adapted, whereas two neighboring populations were not, consistent with a GMC-predicted selection mosaic and a hot spot of adaptation surrounded by areas of maladaptation. Maladaptation was shaped by incongruent Pseudonocardia-Escovopsis population genetic structure, whereas local adaptation was facilitated by geographic isolation on BCI after the flooding of the Panama Canal. Genomic assessments of antibiotic potential of 29 Pseudonocardia strains identified diverse and unique biosynthetic gene clusters in BCI strains despite low genetic diversity in the core genome. The strength of antibiotic inhibition was not correlated with the presence/absence of individual biosynthetic gene clusters or with parasite location. Rather, biosynthetic gene clusters have undergone selective sweeps, suggesting that the trait remixing dynamics conferring the long-term maintenance of antibiotic potency rely on evolutionary genetic changes within already-present biosynthetic gene clusters and not simply on the horizontal acquisition of novel genetic elements or pathways.IMPORTANCE Recently, coevolutionary theory in macroorganisms has been advanced by the geographic mosaic theory of coevolution (GMC), which considers how geography and local adaptation shape coevolutionary dynamics. Here, we test GMC in an ancient symbiosis in which the ant Apterostigma dentigerum cultivates fungi in an agricultural system analogous to human farming. The cultivars are parasitized by the fungus Escovopsis The ants maintain symbiotic actinobacteria with antibiotic properties that help combat Escovopsis infection. This antibiotic symbiosis has persisted for tens of millions of years, raising the question of how antibiotic potency is maintained over these time scales. Our study tests the GMC in a bacterial defensive symbiosis and in a multipartite symbiosis framework. Our results show that this multipartite symbiotic system conforms to the GMC and demonstrate that this theory is applicable in both microbes and indirect symbiont-symbiont interactions.

Keywords: coevolution; geographic mosaic theory of coevolution; secondary metabolism.

FIG 1

The fungus-farming ant Apterostigma dentigerum, seen on top of its fungal garden (A), maintains symbiotic Pseudonocardia actinobacteria, shown here in axenic culture (B). Secondary metabolites from Pseudonocardia inhibit the growth of Escovopsis pathogens that directly consume the fungal garden. Three Escovopsis phylotypes, fuzzy brown (C), yellow (D), and brown (E), are known to infect A. dentigerum gardens. Pseudonocardia and Escovopsis were sampled from Central America (F), with focal populations residing in the Panama Canal Zone (G). Maps created in R using plotly.

FIG 2

(A) Central American Pseudonocardia antibiotic inhibition of three Escovopsis pathogen lineages (fuzzy brown [FZ], yellow, and brown). Rare Escovopsis pathogen morphotypes experience less inhibition by Pseudonocardia (center, median; box, upper and lower quantiles; whiskers, 1.5× interquartile range). (B) Average zone of inhibition (ZOI) corresponds to the relative frequency at which each lineage is encountered in Central American Apterostigma dentigerum ant cultivars, with common brown Escovopsis parasites experiencing significantly greater inhibition than the rarer fuzzy brown and yellow parasites. The strength of inhibition corresponds to parasite morphotype abundance in nature, suggesting that parasites experience rare morph advantage. Blue indicates linear regression of individual data points from panel A error bars indicate standard error of the mean.

FIG 3

A mosaic of antibiotic inhibition by Panamanian Pseudonocardia to Central American populations of Escovopsis (x axis). The average zone of inhibition distance in petri plate bioassays is presented on the y axis (center, median; box, upper and lower quantiles; whiskers, 1.5× interquartile range). Letters indicate significantly different mean zone of inhibition (t test, P < 0.05). LS, La Selva Biological Station; BCI, Barro Colorado Island; PLR, Pipeline Road; GAM, Gamboa Forest.

FIG 4

Reciprocal tests for local adaptation using Pseudonocardia from (A) Barro Colorado Island (BCI), (B) Pipeline Road (PLR), and (C) Gamboa Forest (GAM), with significant local adaptation on BCI indicated by a mean zone of inhibition that was significantly different from the other two pairings in the cross (x axes indicate Escovopsis location). (A to C) t test versus local pairing. *, P = 2.98E−3; **, P = 6.994E−4; ***, P = 8.668E−10; center, median; box, upper and lower quantiles; notches, 95% confidence; whiskers, 1.5× interquartile range; points, outliers. Population STRUCTURE analysis demonstrates that Escovopsis and Pseudonocardia populations on BCI are genetically distinct, having elevated Fk (D) and more than 75% of their isolates assigned to one genetic cluster (E).

FIG 5

Characterization of biosynthetic gene cluster families in symbiotic Pseudonocardia isolates spanning Panama and Costa Rica. (A) Each node represents a contiguous set of genes that are part of a secondary metabolite biosynthesis cluster. Edges represent BiG-SCAPE distances of 0.3 or below. Connected subgraphs correspond to gene cluster families. Nodes are colored by their source (see top right of panel B for legend). (B) Presence-absence map of gene cluster families. Known gene cluster families with examples in MIBiG are shown as dark green in the top annotation strip. Sample location is shown in the left annotation strip. Dendrograms for gene cluster families and samples are derived from Euclidean clustering. (C) Example of selective sweep within a biosynthetic gene cluster. Fst between BCI and other samples is shown as the y axis. Solid and dashed lines correspond to the Fst genome-wide average and two standard deviations above the genome-wide average, respectively (window, 1 kb; step size, 100 bp). A nonribosomal peptide synthetase (NRPS) biosynthetic gene cluster is shown with boundaries corresponding to the shaded region; windows within the gene cluster are shown in purple, and those flanking it are in gray. Gene and domain structure for the biosynthetic gene cluster are shown above. C, condensation domain; A, adenylation domain; TE, thioesterase domain; blue, NRPS genes; pink, TE domain.