The attine ants are a monophyletic lineage that switched to fungus-farming ca. 55-60 MYA. They have become a model for the study of complex symbioses after additional fungal and bacterial symbionts were discovered, but their abdominal endosymbiotic bacteria remain largely unknown. Here we present a comparative microbiome analysis of endosymbiotic bacteria spanning the entire phylogenetic tree. We show that, across 17 representative sympatric species from eight genera sampled in Panama, abdominal microbiomes are dominated by Mollicutes, α- and γ-Proteobacteria, and Actinobacteria. Bacterial abundances increase from basal to crown branches in the phylogeny reflecting a shift towards putative specialized and abundant abdominal microbiota after the ants domesticated gongylidia-bearing cultivars, but before the origin of industrial-scale farming based on leaf-cutting herbivory. This transition coincided with the ancestral single colonization event of Central/North America ca. 20 MYA, documented in a recent phylogenomic study showing that the entire crown-group of the higher attine ants, including the leaf-cutting ants, evolved there and not in South America. Several bacterial species are located in gut tissues or abdominal organs of the evolutionarily derived, but not the basal attine ants. The composition of abdominal microbiomes appears to be affected by the presence/absence of defensive antibiotic-producing actinobacterial biofilms on the worker ants’ cuticle, but the significance of this association remains unclear. The patterns of diversity, abundance, and sensitivity of the abdominal microbiomes that we obtained explore novel territory in the comparative analysis of attine fungus-farming symbioses and raise new questions for further in-depth research.

Understanding connectivity among populations in fragmented landscapes is of paramount importance in species conservation because it determines their long-term viability and helps to identify and prioritize populations to conserve. Rare and sedentary species are particularly vulnerable to habitat fragmentation as they occupy narrow niches or restricted habitat ranges. Here, we assess contemporary interpopulation connectedness of the threatened, myrmecophilous butterfly, Maculinea alcon, in a highly fragmented landscape. We inferred dispersal, effective population sizes, genetic diversity and structure based on 14 locations of M. alcon in Belgium and the Netherlands using data from 12 microsatellite loci. Despite the reported sedentary behaviour of M. alcon, we observed moderate levels of contemporary dispersal between patches, but only in landscapes where populations were located within a distance of 3 km from neighbouring populations. Estimates of effective population sizes (Ne) were very low (ranging from 1.6 to 17.6) and bottleneck events occurred in most of the studied populations. We discuss the functional conservation units delineated based on a former mark-release-recapture study, and formulate appropriate conservation strategies to maintain viable (meta)populations in highly fragmented, anthropogenic landscapes.

Fungus-farming ant colonies vary four to five orders of magnitude in size. They employ compounds from actinomycete bacteria and exocrine glands as antimicrobial agents. Atta colonies have millions of ants and are particularly relevant for understanding hygienic strategies as they have abandoned their ancestors' prime dependence on antibiotic-based biological control in favour of using metapleural gland (MG) chemical secretions. Atta MGs are unique in synthesizing large quantities of phenylacetic acid (PAA), a known but little investigated antimicrobial agent. We show that particularly the smallest workers greatly reduce germination rates of Escovopsis and Metarhizium spores after actively applying PAA to experimental infection targets in garden fragments and transferring the spores to the ants' infrabuccal cavities. In vitro assays further indicated that Escovopsis strains isolated from evolutionarily derived leaf-cutting ants are less sensitive to PAA than strains from phylogenetically more basal fungus-farming ants, consistent with the dynamics of an evolutionary arms race between virulence and control for Escovopsis, but not Metarhizium. Atta ants form larger colonies with more extreme caste differentiation relative to other attines, in societies characterized by an almost complete absence of reproductive conflicts. We hypothesize that these changes are associated with unique evolutionary innovations in chemical pest management that appear robust against selection pressure for resistance by specialized mycopathogens.

Background: The obligate mutualism between fungus-growing ants and microbial symbionts offers excellent opportunities to study the specificity and stability of multi-species interactions. In addition to cultivating fungus gardens, these ants have domesticated actinomycete bacteria to defend gardens against the fungal parasite Escovopsis and possibly other pathogens. Panamanian Acromyrmex echinatior leaf-cutting ants primarily associate with actinomycetes of the genus Pseudonocardia. Colonies are inoculated with one of two vertically transmitted phylotypes (Ps1 or Ps2), and maintain the same phylotype over their lifetime. We performed a cross-fostering experiment to test whether co-adaptations between ants and bacterial phylotypes have evolved, and how this affects bacterial growth and ant prophylactic behavior after infection with Escovopsis. Results: We show that Pseudonocardia readily colonized ants irrespective of their colony of origin, but that the Ps2 phylotype, which was previously shown to be better able to maintain its monocultural integrity after workers became foragers than Ps1, reached a higher final cover when grown on its native host than on alternative hosts. The frequencies of major grooming and weeding behaviors co-varied with symbiont/host combinations, showing that ant behavior also was affected when cuticular actinomycete phylotypes were swapped. Conclusion: These results show that the interactions between leaf-cutting ants and Pseudonocardia bear signatures of mutual co-adaptation within a single ant population.

Ant microgynes are miniaturized queen forms found together with normal queens (macrogynes) in species occurring across the ant phylogeny. Their role is not yet fully understood: in some cases, they seem to be nonparasitic alternative reproductive morphs, in others incipient social parasites, and thus potential models for studying the evolution of social parasitism. Whether they are regarded as parasitic or not has traditionally been based on genetic differentiation from syntopic macrogynes and/or the queen/worker ratio of their offspring rather than measuring fitness traits. We confirmed previously reported genetic differentiation between microgynes and macrogynes of Myrmica rubra in a population studied for the first time. Further, we measured virulence and infectivity of M. rubra microgynes in a controlled laboratory experiment. Nests headed only by macrogynes (controls), only by microgynes, and naturally and artificially mixed nests were kept under identical conditions. We found reduction in worker numbers of both naturally and artificially mixed macrogyne/microgyne nests compared with controls, and strong reduction but also surprising variation in fitness of nests headed only by microgynes. Microgyne nests produced workers, males and microgynes. Microgynes did not themselves reproduce in artificially mixed nests, but reproduced most in naturally mixed nests that had lost their macrogyne queen. This, together with higher mortality of field-collected macrogyne queens from naturally infested colonies and greater estimated relative age of macrogyne queens in naturally infected nests, suggests that they preferentially exploit older host colonies. We conclude that M. rubra microgynes are intraspecific social parasites specialized on exploiting old host colonies.

Wolbachia are renowned as reproductive parasites, but their phenotypic effects in eusocial insects are not well understood. We used a combination of qrt-PCR, fluorescence in situ hybridisation, and laser scanning confocal microscopy to evaluate the dynamics of Wolbachia infections in the leaf-cutting ant Acromyrmex octospinosus across developmental stages of sterile workers. We confirm that workers are infected with one or two widespread wsp genotypes of Wolbachia, show that colony prevalences are always 100%, and characterize two rare recombinant genotypes. One dominant genotype is always present and most abundant while another only proliferates in adult workers of some colonies and is barely detectable in larvae and pupae. An explanation may be that Wolbachia genotypes compete for host resources in immature stages while adult tissues provide substantially more niche space. Tissue-specific prevalences of the two genotypes differ, with the rarer genotype being overrepresented in the adult foregut and thorax muscles. Both genotypes occur extra-cellularly in the foregut, suggesting an unknown mutualistic function in worker ant nutrition. Both genotypes are also abundant in the faecal fluid of the ants, suggesting that they may have extended functional phenotypes in the fungus garden that the ants manure with their own faeces.

Dispersal is crucial for gene flow and often determines the long-term stability of meta-populations, particularly in rare species with specialized life cycles. Such species are often foci of conservation efforts because they suffer disproportionally from degradation and fragmentation of their habitat. However, detailed knowledge of effective gene flow through dispersal is often missing, so that conservation strategies have to be based on mark–recapture observations that are suspected to be poor predictors of long-distance dispersal. These constraints have been especially severe in the study of butterfly populations, where microsatellite markers have been difficult to develop. We used eight microsatellite markers to analyse genetic population structure of the Large Blue butterfly Maculinea arion in Sweden. During recent decades, this species has become an icon of insect conservation after massive decline throughout Europe and extinction in Britain followed by reintroduction of a seed population from the Swedish island of Öland. We find that populations are highly structured genetically, but that gene flow occurs over distances 15 times longer than the maximum distance recorded from mark–recapture studies, which can only be explained by maximum dispersal distances at least twice as large as previously accepted. However, we also find evidence that gaps between sites with suitable habitat exceeding ∼20 km induce genetic erosion that can be detected from bottleneck analyses. Although further work is needed, our results suggest that M. arion can maintain fully functional metapopulations when they consist of optimal habitat patches that are no further apart than ∼10 km.