Specificity of the mutualistic association between actinomycete bacteria and two sympatric species of Acromyrmex leaf‐cutting ants

Abstract

Acromyrmex leaf-cutting ants maintain two highly specialized, vertically transmitted mutualistic ectosymbionts: basidiomycete fungi that are cultivated for food in underground gardens and actinomycete Pseudonocardia bacteria that are reared on the cuticle to produce antibiotics that suppress the growth of Escovopsis parasites of the fungus garden. Mutualism stability has been hypothesized to benefit from genetic uniformity of symbionts, as multiple coexisting strains are expected to compete and, thus, reduce the benefit of the symbiosis. However, the Pseudonocardia symbionts are likely to be involved in Red-Queen-like antagonistic co-evolution with Escovopsis so that multiple strains per host might be favoured by selection provided the cost of competition between bacterial strains is low. We examined the genetic uniformity of the Pseudonocardia symbionts of two sympatric species of Acromyrmex ants by comparing partial sequences of the nuclear Elongation Factor-Tu gene. We find no genetic variation in Pseudonocardia symbionts among nest mate workers, neither in Acromyrmex octospinosus , where colonies are founded by a single queen, nor in Acromyrmex echinatior , where mixing of bacterial lineages might happen when unrelated queens cofound a colony. We further show that the two ant species maintain the same pool of Pseudonocardia symbionts, indicating that horizontal transmission occasionally occurs, and that this pool consists of two distinct clades of closely related Pseudonocardia strains. Our finding that individual colonies cultivate a single actinomycete strain is in agreement with predictions from evolutionary theory on host-symbiont conflict over symbiont mixing, but indicates that there may be constraints on the effectiveness of the bacterial symbionts on an evolutionary timescale.

Specificity in the symbiotic association between fungus-growing ants and protective Pseudonocardia bacteria

“…Cafaro & Currie [27] determined the generic placement of Pseudonocardia using ultrastructure examination of the bacteria directly on the cuticle of Acromyrmex workers. In addition, three population-level studies, representing 180 colonies from four fungusgrowing ant species, support specific associations between fungus-growing ants and Pseudonocardia (34 colonies of Acromyrmex octospinosus and A. echinatior: [28]; 43 colonies in Trachymyrmex septentrionalis: [29]; and 103 colonies in Apterostigma dentigerum: E. J. Caldera 2010, personal communication). Thus, a total of more than 300 colonies of fungus-growing ants have been found to be associated with Pseudonocardia, all of which group phylogenetically within the six clades recognized here (figure 1), supporting that Pseudonocardia is a specific, and potentially the predominant, cuticular exosymbiont.…”

“…Based on their abundance and presence throughout the diversity of the mutualism, and similar findings in previous studies [27][28][29], we included only Pseudonocardia in this study. We obtained Pseudonocardia symbionts from 124 ant colonies, spanning 19 different ant species and broad geographical locations (see electronic supplementary material, table S1).…”

“…Two loci were amplified and sequenced for phylogenetic analyses: the 16S ribosomal gene (1450 bp) using universal bacterial primers (27F and 1492R) and the elongation factor Tu gene (tuf ) (970 bp) using both general (52F and 1048R) and more specific (1F and 920R [28]) bacterial primers. Amplification reactions were standardized to a total final volume between 25 and 50 ml.…”

Generalized antifungal activity and 454-screening ofPseudonocardiaandAmycolatopsisbacteria in nests of fungus-growing ants

“…Most importantly, Pseudonocardia of various attine species have nonspecific antibiotic properties that inhibit garden pathogens, endophytes, saprotrophs, arthropod pathogens, and most severely the ant-cultivated fungi. We evaluate hypotheses on nonadaptive and adaptive functions of attine integumental bacteria and develop an alternative conceptual framework to replace the prevailing Pseudonocardia-Escovopsis model (18)(19)(20)(21)(22)(23)(24)27). For those attine ants for which association with Pseudonocardia is adaptive, possible alternate roles of the integumental bacteria could include protection of the ants, control of cultivar growth, or sanitation of the nest environment.…”

“…This view on specific Pseudonocardia-Escovopsis coevolution was based on very little direct evidence in support of 4 key observations. First, in 2 species studied so far using PCR-based bacterial screens (with Pseudonocardia-specific primers), workers of a single attine nest were thought to associate with only one Pseudonocardia lineage (23). Second, in 2 species studied so far for presence/absence of bacterial growth on reproductives, attine queens carried visible growth during their mating flights, but not the males, suggesting vertical transmission from mother to daughter queen (18); this is expected to generate selection for beneficial bacterial traits within a long-term ant-Pseudonocardia partnership (5,18,20,24).…”